Nogo receptor antagonists

ABSTRACT

Disclosed are immunogenic Nogo receptor-1 polypeptides, Nogo receptor-1 antibodies, antigen-binding fragments thereof, soluble Nogo receptors and fusion proteins thereof and nucleic acids encoding the same. Also disclosed are Nogo receptor antagonist polynucleotides. Also disclosed are compositions comprising, and methods for making and using, such Nogo receptor antibodies, antigen-binding fragments thereof, soluble Nogo receptors and fusion proteins thereof, nucleic acids encoding the same and antagonist polynucleotides.

This application is the National Stage of International Application No.PCT/US2007/002199, filed Jan. 26, 2007, which published under PCTArticle 21(2) in English, which claims the benefit of U.S. ProvisionalApplication 60/762,487, filed Jan. 27, 2006 and U.S. ProvisionalApplication 60/831,659, filed Jul. 19, 2006; all of the aboveapplications are incorporated herein by reference in their entireties.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing (Name:21590970002sequencelisting.txt; Size: 82,764 bytes; and Date ofCreation: May 3, 2012) is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

This invention relates to neurobiology and molecular biology. Moreparticularly, this invention relates to immunogenic Nogo receptor-1(NgR1) polypeptides, Nogo receptor-1 antibodies, antigen-bindingfragments thereof, soluble Nogo receptors and fusion proteins thereofand nucleic acids encoding the same. This invention further relates toNogo receptor-1 antagonist polynucleotides. This invention furtherrelates to compositions comprising, and methods for making and using,such Nogo receptor antibodies, antigen-binding fragments thereof,immunogenic Nogo receptor-1 polypeptides, soluble Nogo receptors andfusion proteins thereof, nucleic acids encoding the same and antagonistpolynucleotides.

BACKGROUND OF THE INVENTION

Axons and dendrites of neurons are long cellular extensions fromneurons. The distal tip of an extending axon or neurite comprises aspecialized region, known as the growth cone. Growth cones sense thelocal environment and guide axonal growth toward the neuron's targetcell. Growth cones respond to several environmental cues, for example,surface adhesiveness, growth factors, neurotransmitters and electricfields. The guidance of growth at the cone involves various classes ofadhesion molecules, intercellular signals, as well as factors thatstimulate and inhibit growth cones. The growth cone of a growing neuriteadvances at various rates, but typically at the speed of one to twomillimeters per day.

Growth cones are hand shaped, with broad flat expansion (microspikes orfilopodia) that differentially adhere to surfaces in the embryo. Thefilopodia are continually active, some filopodia retract back into thegrowth cone, while others continue to elongate through the substratum.The elongations between different filopodia form lamellipodia.

The growth cone explores the area that is ahead of it and on either sidewith its lamellipodia and filopodia. When an elongation contacts asurface that is unfavorable to growth, it withdraws. When an elongationcontacts a favorable growth surface, it continues to extend and guidesthe growth cone in that direction. The growth cone can be guided bysmall variations in surface properties of the substrata. When the growthcone reaches an appropriate target cell a synaptic connection iscreated.

Nerve cell function is greatly influenced by the contact between theneuron and other cells in its immediate environment (U. Rutishauser, T.M. Jessell, Physiol. Rev. 68:819 (1988)). These cells includespecialized glial cells, oligodendrocytes in the central nervous system(CNS), and Schwann cells in the peripheral nervous system (PNS), whichensheathe the neuronal axon with myelin (an insulating structure ofmulti-layered membranes) (G. Lemke, in An Introduction to MolecularNeurobiology, Z. Hall, Ed. (Sinauer, Sunderland, Mass.), p. 281 (1992)).

While CNS neurons have the capacity to regenerate after injury, they areinhibited from doing so because of the presence of inhibitory proteinspresent in myelin and possibly also by other types of molecules normallyfound in their local environment (Brittis and Flanagan, Neuron 30:11-14(2001); Jones et al., J. Neurosc. 22:2792-2803 (2002); Grimpe et al., J.Neurosci. 22:3144-3160 (2002)).

Several myelin inhibitory proteins that are found on oligodendrocyteshave been characterized; e.g., NogoA (Chen et al., Nature 403:434-439(2000); Grandpre et al., Nature 403:439-444 (2000)), myelin associatedglycoprotein (MAG, McKerracher et al., Neuron 13:805-811 (1994);Mukhopadhyay et al., Neuron 13:757-767 (1994)) and oligodendrocyteglycoprotein (OM-gp, Mikol and Stefansson, J. Cell. Biol. 106:1273-1279(1988)). Each of these proteins has been separately shown to be a ligandfor the neuronal Nogo receptor-1 (Wang et al., Nature 417:941-944(2002); Liu et al., Science 297:1190-93 (2002); Grandpre et al., Nature403:439-444 (2000); Chen et al., Nature 403:434-439 (2000); Domeniconiet al., Neuron 35:283-90 (2002)).

Nogo receptor-1 is a GPI-anchored membrane protein that contains 8leucine rich repeats (Fournier et al., Nature 409:341-346 (2001)). Uponinteraction with an inhibitory protein (e.g., NogoA, MAG and OM-gp), theNogo receptor-1 complex transduces signals that lead to growth conecollapse and inhibition of neurite outgrowth.

There is an urgent need for molecules that inhibit Nogo receptor-1binding to its ligands and attenuate myelin-mediated growth conecollapse and inhibition of neurite outgrowth.

SUMMARY OF THE INVENTION

The present invention is directed to the use of Nogo receptor-1antagonists for promoting neurite outgrowth, neuronal survival, andaxonal regeneration in CNS neurons. The invention features molecules andmethods useful for inhibiting neurite outgrowth inhibition, promotingneuronal survival, and/or promoting axonal regeneration in CNS neurons.

The invention also relates to soluble Nogo receptor-1 polypeptides andfusion proteins comprising them, and antibodies and antigenic fragmentsthereof directed against specific immunogenic regions of Nogoreceptor-1. The invention also relates to immunogenic Nogo receptor-1polypeptides that bind to the antibodies of the invention. The inventionalso relates to Nogo receptor-1 polypeptides that are bound by amonoclonal antibody that binds to Nogo receptor-1. Such polypeptides maybe used, inter alia, as immunogens or to screen antibodies to identifythose with similar specificity to an antibody of the invention. Theinvention further relates to nucleic acids encoding the polypeptides ofthis invention, vectors and host cells comprising such nucleic acids andmethods of making the peptides. The antibodies, soluble receptors andreceptor fusion proteins of this invention antagonize or block Nogoreceptor-1 and are useful for inhibiting binding of Nogo receptor-1 toits ligands, inhibiting growth cone collapse in a neuron and decreasingthe inhibition of neurite outgrowth or sprouting in a neuron.

In some embodiments, the invention provides a polypeptide selected fromthe group consisting of AAAFTGLTLLEQLDLSDNAQLR (SEQ ID NO: 26);LDLSDNAQLR (SEQ ID NO: 27); LDLSDDAELR (SEQ ID NO: 29); LDLASDNAQLR (SEQID NO: 30); LDLASDDAELR (SEQ ID NO: 31); LDALSDNAQLR (SEQ ID NO: 32);LDALSDDAELR (SEQ ID NO: 33); LDLSSDNAQLR (SEQ ID NO: 34); LDLSSDEAELR(SEQ ID NO: 35); DNAQLRVVDPTT (SEQ ID NO: 36); DNAQLR (SEQ ID NO: 37);ADLSDNAQLRVVDPTT (SEQ ID NO: 41); LALSDNAQLRVVDPTT (SEQ ID NO: 42);LDLSDNAALRVVDPTT (SEQ ID NO: 43); LDLSDNAQLHVVDPTT (SEQ ID NO: 44); andLDLSDNAQLAVVDPTT (SEQ ID NO: 45).

In some embodiments, the invention provides a nucleic acid encoding apolypeptide of the invention. In some embodiments, the nucleic acid isoperably linked to an expression control sequence. In some embodiments,the invention provides a vector comprising a nucleic acid of theinvention.

In some embodiments, the invention provides a host cell comprising anucleic acid or comprising the vector of the invention. In someembodiments, the invention provides a method of producing a polypeptideof the invention comprising culturing a host cell comprising a nucleicacid or vector of the invention and recovering the polypeptide from thehost cell or culture medium.

In some embodiments, the invention provides a method of producing anantibody comprising the steps of immunizing a host with a polypeptide ofthe invention or a host cell comprising a nucleic acid or comprising thevector of the invention and recovering the antibody. The invention alsoprovides an antibody produced by the method or an antigen-bindingfragment thereof.

In some embodiments, the invention provides an antibody or anantigen-binding fragment thereof that specifically binds to apolypeptide of the invention, wherein the antibody is not the monoclonalantibody produced by hybridoma cell line HB 7E11 (ATCC® accession No.PTA4587).

In some embodiments of the invention, the antibody or antigen-bindingfragment (a) inhibits growth cone collapse of a neuron; (b) decreasesthe inhibition of neurite outgrowth and sprouting in a neuron; and (c)inhibits Nogo receptor-1 binding to a ligand. In some embodiments, theneurite outgrowth and sprouting is axonal growth. In some embodiments,the neuron is a central nervous system neuron.

In some embodiments, an antibody of the invention is monoclonal. In someembodiments, an antibody of the invention is a murine antibody. In someembodiments, an antibody of the invention is selected from the groupconsisting of a humanized antibody, a chimeric antibody and a singlechain antibody.

In some embodiments, the invention provides a method of inhibiting Nogoreceptor-1 binding to a ligand, comprising the step of contacting Nogoreceptor-1 with an antibody of the invention or antigen-binding fragmentthereof. In some embodiments the ligand is selected from the groupconsisting of NogoA, NogoB, NogoC, MAG and OM-gp.

In some embodiments, the invention provides a method for inhibitinggrowth cone collapse in a neuron, comprising the step of contacting theneuron with an antibody of the invention or antigen-binding fragmentthereof.

In some embodiments, the invention provides a method for decreasing theinhibition of neurite outgrowth or sprouting in a neuron, comprising thestep of contacting the neuron with an antibody of the invention orantigen-binding fragment thereof. In some embodiments, the neuriteoutgrowth or sprouting is axonal growth. In some embodiments, the neuronis a central nervous system neuron.

In some embodiments, the invention provides a composition comprising apharmaceutically acceptable carrier and an antibody of the invention oran antigen-binding fragment thereof. In some embodiments, thecomposition further comprises one or more additional therapeutic agents.

In some embodiments, the invention provides a method of promotingsurvival of a neuron at risk of dying, comprising contacting the neuronwith an effective amount of an anti-Nogo receptor-1 antibody of theinvention or antigen-binding fragment thereof. In some embodiments, theneuron is in vitro. In some embodiments, the neuron is in a mammal. Insome embodiments, the mammal displays signs or symptoms of multiplesclerosis, ALS, Huntington's disease, Alzheimer's disease, Parkinson'sdisease, diabetic neuropathy, stroke, traumatic brain injuries or spinalcord injury.

In some embodiments, the invention provides a method of promotingsurvival of a neuron in a mammal, which neuron is at risk of dying,comprising (a) providing a cultured host cell expressing an anti-Nogoreceptor-1 antibody of the invention or antigen-binding fragmentthereof; and (b) introducing the host cell into the mammal at or nearthe site of the neuron.

In some embodiments, the invention provides a gene therapy method ofpromoting survival of a neuron at risk of dying, which neuron is in amammal, comprising administering at or near the site of the neuron aviral vector comprising a nucleotide sequence that encodes an anti-Nogoreceptor-1 antibody of the invention or an antigen-binding fragmentthereof, wherein the anti-Nogo receptor-1 antibody or antigen-bindingfragment is expressed from the nucleotide sequence in the mammal in anamount sufficient to promote survival of the neuron.

In some embodiments, the invention provides an isolated polypeptide of60 residues or less comprising an amino acid sequence selected from thegroup consisting of: amino acids 309 to 335 of SEQ ID NO:49; amino acids309 to 336 of SEQ ID NO:49; amino acids 309 to 337 of SEQ ID NO:49;amino acids 309 to 338 of SEQ ID NO:49; amino acids 309 to 339 of SEQ IDNO:49; amino acids 309 to 340 of SEQ ID NO:49; amino acids 309 to 341 ofSEQ ID NO:49; amino acids 309 to 342 of SEQ ID NO:49; amino acids 309 to343 of SEQ ID NO:49; amino acids 309 to 344 of SEQ ID NO:49; amino acids309 to 345 of SEQ ID NO:49; amino acids 309 to 346 of SEQ ID NO:49;amino acids 309 to 347 of SEQ ID NO:49; amino acids 309 to 348 of SEQ IDNO:49; amino acids 309 to 349 of SEQ ID NO:49; amino acids 309 to 350 ofSEQ ID NO:49; amino acids 300 to 344 of SEQ ID NO:49; amino acids 301 to344 of SEQ ID NO:49; amino acids 302 to 344 of SEQ ID NO:49; amino acids303 to 344 of SEQ ID NO:49; amino acids 304 to 344 of SEQ ID NO:49;amino acids 305 to 344 of SEQ ID NO:49; amino acids 306 to 344 of SEQ IDNO:49; amino acids 307 to 344 of SEQ ID NO:49; amino acids 308 to 344 ofSEQ ID NO:49; amino acids 336 to 344 of SEQ ID NO:49; amino acids 335 to344 of SEQ ID NO:49; amino acids 334 to 344 of SEQ ID NO:49; amino acids333 to 344 of SEQ ID NO:49; amino acids 332 to 344 of SEQ ID NO:49;amino acids 331 to 344 of SEQ ID NO:49; amino acids 330 to 344 of SEQ IDNO:49; amino acids 329 to 344 of SEQ ID NO:49; amino acids 328 to 344 ofSEQ ID NO:49; amino acids 327 to 344 of SEQ ID NO:49; amino acids 326 to344 of SEQ ID NO:49; amino acids 325 to 344 of SEQ ID NO:49; amino acids324 to 344 of SEQ ID NO:49; amino acids 323 to 344 of SEQ ID NO:49;amino acids 322 to 344 of SEQ ID NO:49; amino acids 321 to 344 of SEQ IDNO:49; amino acids 320 to 344 of SEQ ID NO:49; amino acids 319 to 344 ofSEQ ID NO:49; amino acids 318 to 344 of SEQ ID NO:49; amino acids 317 to344 of SEQ ID NO:49; amino acids 316 to 344 of SEQ ID NO:49; amino acids315 to 344 of SEQ ID NO:49; amino acids 314 to 344 of SEQ ID NO:49;amino acids 313 to 344 of SEQ ID NO:49; amino acids 312 to 344 of SEQ IDNO:49; amino acids 311 to 344 of SEQ ID NO:49; amino acids 310 to 344 ofSEQ ID NO:49; amino acids 336 to 344 of SEQ ID NO:49; amino acids 336 to345 of SEQ ID NO:49; amino acids 336 to 346 of SEQ ID NO:49; amino acids336 to 347 of SEQ ID NO:49; amino acids 336 to 348 of SEQ ID NO:49;amino acids 336 to 349 of SEQ ID NO:49; amino acids 336 to 350 of SEQ IDNO:49; variants or derivatives of any of said polypeptide fragments, anda combination of at least two of any of said polypeptide fragments;except for up to three amino acid substitutions.

In some embodiments, the invention provides an isolated polypeptide of60 residues or less comprising an amino acid sequence selected from thegroup consisting of: amino acids 311-344 of SEQ ID NO:49; amino acids310-348 of SEQ ID NO:49; amino acids 323-328 of SEQ ID NO:49; aminoacids 339-348 of SEQ ID NO:49; amino acids 378-414 of SEQ ID NO:49;amino acids 27-38 of SEQ ID NO:49; amino acids 39-61 of SEQ ID NO:49;amino acids 257-267 of SEQ ID NO:49; amino acids 280-292 of SEQ IDNO:49; amino acids 301-323 of SEQ ID NO:49; amino acids 335-343 of SEQID NO:49; amino acids 310-335 of SEQ ID NO:49; amino acids 326-328 ofSEQ ID NO:49; variants or derivatives of any of said polypeptidefragments, and a combination of at least two of any of said polypeptidefragments.

In some embodiments, the invention provides an isolated polypeptidefragment of 60 residues or less, comprising an amino acid sequenceidentical to a reference amino acid sequence, except for up to threeindividual amino acid substitutions, wherein said reference amino acidsequence is selected from the group consisting of: (a) amino acids x to344 of SEQ ID NO:49, (b) amino acids 309 to y of SEQ ID NO:49, and (c)amino acids x to y of SEQ ID NO:49, wherein x is any integer from 305 to326, and y is any integer from 328 to 350; and wherein said polypeptidefragment inhibits Nogo-receptor-mediated neurite outgrowth inhibition.In some embodiments, the invention provides an isolated polypeptidefragment of 60 residues or less, comprising an amino acid sequenceidentical to a reference amino acid sequence, except for up to threeindividual amino acid substitutions, wherein said reference amino acidsequence is selected from the group consisting of: (a) amino acids x′ to344 of SEQ ID NO:49, (b) amino acids 309 to y′ of SEQ ID NO:49, and (c)amino acids x′ to y′ of SEQ ID NO:49, where x′ is any integer from 300to 326, and y′ is any integer from 328 to 360, and wherein saidpolypeptide fragment inhibits Nogo-receptor-mediated neurite outgrowthinhibition. In some embodiments, the invention provides an isolatedpolypeptide fragment of 60 residues or less, comprising an amino acidsequence identical to a reference amino acid sequence, except for up tothree individual amino acid substitutions, wherein said reference aminoacid sequence is selected from the group consisting of: amino acids 309to 335 of SEQ ID NO:49; amino acids 309 to 344 of SEQ ID NO:49; aminoacids 310 to 335 of SEQ ID NO:49; amino acids 310 to 344 of SEQ IDNO:49; amino acids 309 to 350 of SEQ ID NO:49; amino acids 300 to 344 ofSEQ ID NO:49; and amino acids 315 to 344 of SEQ ID NO:49. In someembodiments, the invention provides an isolated polypeptide fragment of60 residues or less, comprising an amino acid sequence identical to areference amino acid sequence, except for up to three individual aminoacid substitutions, wherein said reference amino acid sequence is aminoacids 309 to 344 of SEQ ID NO:49. In some embodiments, the inventionprovides an isolated polypeptide fragment of 60 residues or less,comprising an amino acid sequence identical to a reference amino acidsequence, except for up to three individual amino acid substitutions,wherein said reference amino acid sequence is amino acids 309 to 335 ofSEQ ID NO:49.

In some embodiments, the invention provides a polypeptide of theinvention that is cyclic. In some embodiments, the cyclic polypeptidefurther comprises a first molecule linked at the N-terminus and a secondmolecule linked at the C-terminus; wherein the first molecule and thesecond molecule are joined to each other to form said cyclic molecule.In some embodiments, the first and second molecules are selected fromthe group consisting of: a biotin molecule, a cysteine residue, and anacetylated cysteine residue. In some embodiments, the first molecule isa biotin molecule attached to the N-terminus and the second molecule isa cysteine residue attached to the C-terminus of the polypeptide of theinvention. In some embodiments, the first molecule is an acetylatedcysteine residue attached to the N-terminus and the second molecule is acysteine residue attached to the C-terminus of the polypeptide of theinvention. In some embodiments, the first molecule is an acetylatedcysteine residue attached to the N-terminus and the second molecule is acysteine residue attached to the C-terminus of the polypeptide of theinvention. In some embodiments, the C-terminal cysteine has an NH2moiety attached.

In some embodiments, the invention provides an isolated polypeptidecomprising a first polypeptide fragment and a second polypeptidefragment, wherein said first polypeptide fragment comprises an aminoacid sequence identical to a first reference amino acid sequence, exceptfor up to twenty individual amino acid substitutions, wherein said firstreference amino acid sequence is selected from the group consisting of:(a) amino acids a to 445 of SEQ ID NO:49, (b) amino acids 27 to b of SEQID NO:49, and (c) amino acids a to b of SEQ ID NO:49, wherein a is anyinteger from 25 to 35, and b is any integer from 300 to 450; whereinsaid second polypeptide fragment comprises an amino acid sequenceidentical to a second reference amino acid sequence, except for up totwenty individual amino acid substitutions, wherein said secondreference amino acid sequence is selected from the group consisting of(a) amino acids c to 445 of SEQ ID NO:49, (b) amino acids 27 to d of SEQID NO:49, and (c) amino acids c to d of SEQ ID NO:49, wherein c is anyinteger from 25 to 35, and d is any integer from 300 to 450; and whereinsaid polypeptide inhibits Nogo-receptor-mediated neurite outgrowthinhibition. In some embodiments, the invention further provides anisolated polypeptide comprising a first polypeptide fragment and asecond polypeptide fragment, wherein the first polypeptide fragmentcomprises an amino acid sequence identical to a first reference aminoacid sequence, except for up to twenty individual amino acidsubstitutions, wherein the first reference amino acid sequence isselected from the group consisting of: (a) amino acids 27 to 310 of SEQID NO:49 and (b) amino acids 27 to 344 of SEQ ID NO:49. In someembodiments, the invention further provides an isolated polypeptidecomprising a first polypeptide fragment and a second polypeptidefragment, wherein the second polypeptide fragment comprises an aminoacid sequence identical to a first reference amino acid sequence, exceptfor up to twenty individual amino acid substitutions, wherein the secondreference amino acid sequence is selected from the group consisting of:(a) amino acids 27 to 310 of SEQ ID NO:49 and (b) amino acids 27 to 344of SEQ ID NO:49. In some embodiments, the invention further provides anisolated polypeptide comprising a first polypeptide fragment and asecond polypeptide fragment, wherein the second polypeptide fragmentcomprises an amino acid sequence identical to a first reference aminoacid sequence, except for up to twenty individual amino acidsubstitutions, wherein the first polypeptide fragment comprises aminoacids 27 to 310 of SEQ ID NO:49 and the second polypeptide fragmentcomprises amino acids 27 to 310 of SEQ ID NO:49 or wherein the firstpolypeptide fragment comprises amino acids 27 to 344 of SEQ ID NO:49 andthe second polypeptide fragment comprises amino acids 27 to 310 of SEQID NO:49 or wherein the first polypeptide fragment comprises amino acids27 to 344 of SEQ ID NO:49 and the second polypeptide fragment comprisesamino acids 27 to 344 of SEQ ID NO:49. In some embodiments, theinvention further provides that the first polypeptide fragment issituated upstream of the second polypeptide fragment. In someembodiments, the invention further provides a peptide linker situatedbetween the first polypeptide fragment and the second polypeptidefragment. In some embodiments, the invention further provides that thepeptide linker comprises SEQ ID NO:65 (G4S)₃. In some embodiments, theinvention further provides that the peptide linker comprises SEQ IDNO:66 (G4S)₂.

In some embodiments, the invention provides a polypeptide of theinvention wherein at least one cysteine residue is substituted with aheterologous amino acid. In some embodiments, the at least one cysteineresidue is C266. In some embodiments, the at least one cysteine residueis C309. In some embodiments, the at least one cysteine residue is C335.In some embodiments, the at least one cysteine residue is at C336. Insome embodiments, the at least one cysteine residue is substituted witha replacement amino acid selected from the group consisting of alanine,serine and threonine. In some embodiments, the replacement amino acid isalanine.

In some embodiments, the invention provides an isolated polypeptidecomprising: (a) an amino acid sequence identical to a reference aminoacid sequence except that at least one cysteine residue of saidreference amino acid sequence is substituted with a different aminoacid, wherein said reference amino acid sequence is selected from thegroup consisting of: (i) amino acids a to 445 of SEQ ID NO:49, (iii)amino acids 27 to b of SEQ ID NO:49, and (iii) amino acids a to b of SEQID NO:49, wherein a is any integer from 25 to 35, and b is any integerfrom 300 to 450; and (b) a heterologous polypeptide; wherein saidpolypeptide inhibits nogo-receptor-mediated neurite outgrowthinhibition. In some embodiments, the invention further provides thatC266 of said reference amino acid sequence is substituted with adifferent amino acid. In some embodiments, the invention furtherprovides that C309 of said reference amino acid sequence is substitutedwith a different amino acid. In some embodiments, the invention furtherprovides that C335 of said reference amino acid sequence is substitutedwith a different amino acid. In some embodiments, the invention furtherprovides that C266 and C309 of said reference amino acid sequence aresubstituted with different amino acids. In some embodiments, theinvention further provides that C309 and C335 of said reference aminoacid sequence are substituted with different amino acids. In someembodiments, the invention further provides that the different aminoacid is alanine.

In some embodiments, the invention provides an isolated polypeptidecomprising: (a) an amino acid sequence identical to a reference aminoacid sequence, except for up to twenty individual amino acidsubstitutions, wherein said reference amino acid sequence is selectedfrom the group consisting of: (i) amino acids a to 445 of SEQ ID NO:49,(ii) amino acids 27 to b of SEQ ID NO:49, and (iii) amino acids a to bof SEQ ID NO:49, wherein a is any integer from 25 to 35, and b is anyinteger from 300 to 450; and (b) a heterologous polypeptide; whereinsaid polypeptide inhibits nogo-receptor-mediated neurite outgrowthinhibition.

In some embodiments, the invention provides an isolated polypeptidecomprising a first polypeptide fragment and a second polypeptidefragment, wherein said first polypeptide fragment comprises an aminoacid sequence identical to a first reference amino acid sequence, exceptfor up to twenty individual amino acid substitutions, wherein said firstreference amino acid sequence is selected from the group consisting of:(a) amino acids a to 305 of SEQ ID NO:49, (b) amino acids 1 to b of SEQID NO:49, and (c) amino acids a to b of SEQ ID NO:49, wherein a is anyinteger from 1 to 27, and b is any integer from 264 to 309; and whereinsaid second polypeptide fragment comprises an amino acid sequenceidentical to a second reference amino acid sequence, except for up totwenty individual amino acid substitutions, wherein said secondreference amino acid sequence is selected from the group consisting of:(a) amino acids c to 332 of SEQ ID NO:60, (b) amino acids 275 to d ofSEQ ID NO:60, and (c) amino acids c to d of SEQ ID NO:60, wherein c isany integer from 265 to 306, and d is any integer from 308 to 340; andwherein said polypeptide inhibits nogo-receptor-mediated neuriteoutgrowth inhibition. In certain embodiments, the first reference aminoacid sequence is selected from the group consisting of: (a) amino acids1-274 of SEQ ID NO:49; and (b) amino acids 1-305 of SEQ ID NO:49. Incertain embodiments, the second reference amino acid sequence isselected from the group consisting of: (a) amino acids 275-311 of SEQ IDNO:60; (b) amino acids 275-332 of SEQ ID NO:60; (c) amino acids 306-311of SEQ ID NO:60; (d) amino acids 306-308 of SEQ ID NO:60; and (e) aminoacids 306-309 of SEQ ID NO:60. In one embodiment, the first polypeptidefragment comprises amino acids 1-274 of SEQ ID NO:49 and the secondpolypeptide fragment comprises amino acids amino acids 275-311 of SEQ IDNO:60. In some embodiments, the first polypeptide fragment comprisesamino acids 1-274 of SEQ ID NO:49 and the second polypeptide fragmentcomprises amino acids 275-332 of SEQ ID NO:60. In some embodiments, thefirst polypeptide fragment comprises amino acids 1-305 of SEQ ID NO:49and the second polypeptide fragment comprises amino acids 306-311 of SEQID NO:60. In some embodiments, the first polypeptide fragment comprisesamino acids 1-305 of SEQ ID NO:49 and the second polypeptide fragmentcomprises amino acids 306-308 of SEQ ID NO:60. In some embodiments, thefirst polypeptide fragment comprises amino acids 1-305 of SEQ ID NO:49and the second polypeptide fragment comprises amino acids 306-309 of SEQID NO:60. In some embodiments at least one additional amino acid isadded to the C-terminus of the second polypeptide fragment. In oneembodiment, the at least one additional amino acid is tryptophan. Insome embodiments, A269 of the first polypeptide fragment is substitutedwith a different amino acid. In one embodiment, the different amino acidis tryptophan.

In some embodiments the invention provides an isolated polypeptidecomprising a first polypeptide fragment and a second polypeptidefragment, wherein said first polypeptide fragment consists of aminoacids 1-310 of SEQ ID NO:49, except for up to twenty individual aminoacid substitutions; and wherein said second polypeptide fragmentconsists of amino acids 311 to 318 of SEQ ID NO:60, except for up tofive individual amino acid substitutions; and wherein said polypeptideinhibits nogo-receptor-mediated neurite outgrowth inhibition.

In some embodiments the invention further provides that the heterologouspolypeptide is selected from the group consisting of: (a) serum albumin,(b) an Fc region, (c) a signal peptide, (d) a polypeptide tag, and (e) acombination of two or more of said heterologous polypeptides. In someembodiments, the invention further provides that the Fc region isselected from the group consisting of: an IgA Fe region; an IgD Feregion; an IgG Fe region, an IgEFc region; and an IgM Fe region. In oneembodiment, the Fc region is an IgG Fc region. In some embodiments, theinvention further provides that a peptide linker is situated between theamino acid sequence and the IgG Fc region. In one embodiment, thepeptide linker comprises SEQ ID NO:66 (G4S)₂. In some embodiments, theinvention further provides that the polypeptide tag is selected from thegroup consisting of: FLAG tag; Strep tag; poly-histidine tag; VSV-G tag;influenza virus hemagglutinin (HA) tag; and c-Myc tag.

In some embodiments, the invention provides a polypeptide of theinvention attached to one or more polyalkylene glycol moieties. In someembodiments, the invention further provides that the one or morepolyalkylene glycol moieties is a polyethylene glycol (PEG) moiety. Insome embodiments, the invention further provides a polypeptide of theinvention attached to 1 to 5 PEG moieties.

In some embodiments, the invention provides an isolated polynucleotideencoding a polypeptide of the invention. In some embodiments, theinvention further provides that the nucleotide sequence is operablylinked to an expression control element (e.g. an inducible promoter, aconstitutive promoter, or a secretion signal). Additional embodimentsinclude a vector comprising an isolated polynucleotide of the inventionand a host cell comprising said vector.

In some embodiments, the invention provides an isolated polynucleotideselected from the group consisting of: (i) an antisense polynucleotide;(ii) a ribozyme; (iii) a small interfering RNA (siRNA); and (iv) asmall-hairpin RNA (shRNA).

In some embodiments, the isolated polynucleotide is an antisensepolynucleotide comprising at least 10 bases complementary to the codingportion of the NgR1 mRNA. In some embodiments, the polynucleotide is aribozyme.

In further embodiments, the polynucleotide is a siRNA or a shRNA. Insome embodiments, the invention provides that that siRNA or the shRNAinhibits NgR1 expression. In some embodiments, the invention furtherprovides that the siRNA or shRNA is at least 90% identical to thenucleotide sequence comprising: CUACUUCUCCCGCAGGCG orCCCGGACCGACGUCUUCAA or CUGACCACUGAGUCUUCCG. In other embodiments, thesiRNA or shRNA nucleotide sequence is CUACUUCUCCCGCAGGCG orCCCGGACCGACGUCUUCAA or CUGACCACUGAGUCUUCCG.

In some embodiments, the invention further provides that the siRNA orshRNA nucleotide sequence is complementary to the mRNA produced by thepolynucleotide sequence GATGAAGAGGGCGTCC GCT or GGGCCTGGCTGCAGAAGTT orGACTGGTGACTCAGAG AAGGC.

Additional embodiments of the invention include pharmaceuticalcompositions comprising the polypeptides, polynucleotides, vectors orhost cells of the invention and in certain embodiments apharmaceutically acceptable carrier. In certain embodiments, thecomposition comprises amino acids 27-310 of SEQ ID NO: 7 and ananti-inflammatory agent. In other embodiments, the composition comprisesamino acids 27-310 of SEQ ID NO: 9 and an anti-inflammatory agent. Insome embodiments, the invention further provides that the inflammatoryagent is selected from the group consisting of a steroidalanti-inflammatory agent and a non-steroidal anti-inflammatory agent. Incertain embodiments, the steroidal anti-inflammatory agent is selectedfrom the group consisting of hydrocortisone, 21-acetoxypregnenolone,alclomerasone, algestone, amcinonide, beclomethasone, betamethasone,betamethasone valerate, budesonide, chloroprednisone, clobetasol,clobetasol propionate, clobetasone, clobetasone butyrate, clocortolone,cloprednol, corticosterone, cortisone, cortivazol, deflazacon, desonide,desoximerasone, dexamethasone, diflorasone, diflucortolone,difluprednate, enoxolone, fluazacort, flucloronide, flumethasone,flumethasone pivalate, flunisolide, flucinolone acetonide, fluocinonide,fluorocinolone acetonide, fluocortin butyl, fluocortolone,fluorocortolone hexanoate, diflucortolone valerate, fluorometholone,fluperolone acetate, fluprednidene acetate, fluprednisolone,flurandenolide, formocortal, halcinonide, halometasone, halopredoneacetate, hydrocortamate, hydrocortisone, hydrocortisone acetate,hydrocortisone butyrate, hydrocortisone phosphate, hydrocortisone21-sodium succinate, hydrocortisone tebutate, mazipredone, medrysone,meprednisone, methylprednisolone, mometasone furoate, paramethasone,prednicarbate, prednisolone, prednisolone 21-diedryaminoacetate,prednisolone sodium phosphate, prednisolone sodium succinate,prednisolone sodium 21-m-sulfobenzoate, prednisolone sodium21-stearoglycolate, prednisolone tebutate, prednisolone21-trimethylacetate, prednisone, prednival, prednylidene, prednylidene21-diethylaminoacetate, tixocortol, triamoinolone, triamcinolone,acetonide, triamcinolone benetonide and triamcinolone hexacetonide. In aparticular embodiment, the steroidal anti-inflammatory agent ismethylprednisolone. In other embodiments, the non-steroidalanti-inflammatory agent is selected from the group consisting ofalminoprofen, benoxaprofen, bucloxic acid, carprofen, fenbufen,fenoprofen, fluprofen, flurbiprofen, ibuprofen, indoprofen, ketoprofen,miroprofen, naproxen, oxaprozin, pirprofen, pranoprofen, suprofen,tiaprofenic acid, tioxaprofen, indomethacin, acemetacin, alclofenac,clidanac, diclofenac, fenclofenac, fenclozic acid, fentiazac, furofenac,ibufenac, isoxepac, oxpinac, sulindac, tiopinac, tolmetin, zidometacin,zomepirac, flufenamic acid, meclofenamic acid, mefenamic acid, niflumicacid, tolfenamic acid, diflunisal, flufenisal, isoxicam, piroxicam,sudoxicam, tenoxicam, acetyl salicylic acid, sulfasalazine, apazone,bezpiperylon, feprazone, mofebutazone, oxyphenbutazone andphenylbutazone.

Additional embodiments include compositions where amino acids 27-310 ofSEQ ID NO: 7 or 9 are fused to a heterologous polypeptide. In someembodiments, the heterologous polypeptide is Fc.

Embodiments of the invention also include methods for promoting neuriteoutgrowth, comprising contacting a neuron with an agent which includespolypeptides, polynucleotides or compositions of the invention, whereinsaid agent inhibits Nogo receptor 1-mediated neurite outgrowthinhibition.

Additional embodiments include a method for inhibiting signaltransduction by the NgR1 signaling complex, comprising contacting aneuron with an effective amount of an agent which includes polypeptide,polynucleotides, or compositions of the invention, wherein said agentinhibits signal transduction by the NgR1 signaling complex.

Other embodiments include a method for treating a central nervous system(CNS) disease, disorder, or injury in a mammal, comprising administeringto a mammal in need of treatment an effective amount of an agent whichincludes polypeptides, polynucleotides, or compositions of theinvention, wherein said agent inhibits Nogo Receptor 1-mediated neuriteoutgrowth inhibition. In certain embodiments, the disease, disorder orinjury is selected from the group consisting of multiple sclerosis, ALS,Huntington's disease, Alzheimer's disease, Parkinson's disease, diabeticneuropathy, stroke, traumatic brain injuries, spinal cord injury, opticneuritis, glaucoma, hearing loss, and adrenal leukodystrophy.

In some embodiments the invention further provides that the polypeptideis fused to a heterologous polypeptide. In some embodiments, theheterologous polypeptide is serum albumin. In some embodiments, theheterologous polypeptide is an Fc region. In some embodiments, theheterologous polypeptide is a signal peptide. In some embodiments, theheterologous polypeptide is a polypeptide tag. In some embodiments, theinvention further provides that the Fc region is selected from the groupconsisting of: an IgA Fc region; an IgD Fc region; an IgG Fc region, anIgEFc region; and an IgM Fc region. In some embodiments, the inventionfurther provides that the polypeptide tag is selected from the groupconsisting of: FLAG tag; Strep tag; poly-histidine tag; VSV-G tag;influenza virus hemagglutinin (HA) tag; and c-Myc tag.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the structure of Nogoreceptor-1. Human sNogoR310 contains residues 26-310 of SEQ ID NO:7 andsNogoR344 contains residues 26-344 of SEQ ID NO:6. Rat sNogoR310contains residues 27-310 of SEQ ID NO:9 and sNogoR344 contains residues27-344 of SEQ ID NO:8.

FIG. 2 depicts an antigenicity plot for the Nogo receptor-1 proteinusing the Vector Nti™ software. Rat P-617 is SEQ ID NO: 10 and rat P-618is SEQ ID NO: 11. Human P-617 is SEQ ID NO:47 and human P-618 is SEQ IDNO:48.

FIG. 3A is a graph depicting the binding activity of anti-Nogoreceptor-1 antibody, 7E11. The graph presents the effect of 7E11concentration on the binding of Nogo66 to Nogo receptor-1. FIG. 3Bdepicts the binding activity of anti-Nogo receptor-1 antibody, 1H2. Thegraph presents the effect of 1H2 concentration on the binding of Nogo66to sNogoR344-Fc (also referred to herein and in U.S. patent application60/402,866 as Fc-sNogoR344 or Ig-sNogoR344). Fc-MAG did not compete withNogo66 for binding to sNogoR344-Fc.

FIG. 4 depicts the results of an ELISA for anti-Nogo-R-1 antibodies 1H2,3G5 and 2F7. The effect of the antibodies on OD₄₅₀ in the presence ofimmobilized antigens was determined. The immobilized antigens weresNogoR310-Fc (also referred to herein and in U.S. patent application60/402,866 as Fc-sNogoR310 or Ig-sNogoR310), sNogoR344-Fc, p-617, p-618,p4, p-5 and ovalbumin and BSA.

FIG. 5 is a graph depicting the effects of monoclonal antibody, 7E11, onrat DRG neurite outgrowth in the presence of varying amounts of myelin.

FIG. 6A is a graph depicting the effect of binding of sNogoR310 to¹²⁵I-Nogo66 and ¹²⁵I-Nogo40 in the presence of the followingcompetitors: Nogo66, Nogo40 and anti-Nogo receptor-1 monoclonal antibodysupernatant. FIG. 6B depicts the binding activity of ¹²⁵I-Nogo66 tosNogoR310.

FIG. 7 is a graph depicting the effect of sNogoR310-Fc on ¹²⁵I-Nogo40binding to sNogoR310.

FIG. 8 is a graph depicting the binding activity of sNogoR310-Fc to¹²⁵I-Nogo40.

FIG. 9A is a graph of the effect of sNogoR310 on neurite outgrowth/cellin the presence or absence of myelin. FIG. 9B is a graph of the effectof sNogoR310 on neurite outgrowth in the presence or absence of myelin.

FIG. 10A is a graph depicting the effect of sNogoR310-Fc on P4 rat DRGneurite outgrowth in the presence or absence of increasing amounts ofmyelin. FIG. 10B depicts the number of neurites/cell following treatmentwith PBS, PBS+sNogoR310-Fc, 20 ng myelin and myelin+sNogoR310-Fc.

FIG. 11 is a graph depicting the effect of monoclonal antibody 5B10 onDRG neurite outgrowth/cell in the presence of increasing amounts ofmyelin.

FIG. 12 is a graph depicting the effect of sNogoR310-Fc on the BBB scoreup to 30 days following induction of injury in a rat spinal cordtransection model.

FIGS. 13A and 13B report the locomotor BBB score as a function of timeafter dorsal hemisection in the WT or transgenic mice from Line 08 orLine 01. FIG. 13C graphs the maximal tolerated inclined plane angle as afunction of time after injury for WT and transgenic mice. FIG. 13D showshindlimb errors during inclined grid climbing as a function ofpost-injury time. In all the graphs, means±s.e.m. from 7-9 mice in eachgroup are reported. The values from transgenic group are statisticallydifferent from the WT mice. (double asterisks, P<0.01; Student'st-test).

FIG. 14A shows the locomotor BBB score as a function of time afterdorsal hemisection in vehicle or sNogoR310-Fc treated animals. FIG. 14Bshows hindlimb errors during grid walking as a function of time afterinjury. FIG. 14C shows footprint analysis revealing a shorter stridelength and a greater stride width in control mice than uninjured orinjured+sNogoR310-Fc rats. In all the graphs, means±s.e.m. from 7-9 ratsin each group are reported. The values of sNogoR310-Fc group arestatistically different from the control (FIGS. 14A-B). The controlvalues are statistically different from no-SCI or SCI+sNogoR310-Fc ratsin FIG. 14C. (asterisk, p<0.05; double asterisks, p<0.01; Student'st-test).

FIG. 15 shows a model of the binding of the anti-rNgR1 antibody, 1D9, tothe soluble fragment of rat NgR1 (srNgR310).

FIG. 16A shows the nucleotide sequence of human Nogo receptor cDNA. Thestart and stop codons are underlined. The selected RNAi target regionsare italicized. RNAi-1 and RNAi-3 target the human NgR genespecifically, RNAi-2 was designed to target human, mouse and rat NgRgenes. FIG. 16 B shows the nucleotide sequences of the DNAoligonucleotides used for construction of NgR RNAi into expressionvector pU6.

FIG. 17 depicts the results of the transient transfection test of RNAiknockdown in mouse L cells.

FIG. 18 shows the transfection of the human NgR expression vector intoSKN and 293 cells.

FIG. 19 depicts the transient transfection test of RNAi knockdown inhuman SKN cells.

FIG. 20 shows a schematic representation of a RNAi lentiviral vector.The RNAi expression cassette can be inserted at the PacI site or EcoRIsite. LTR-long terminal repeat; RRE-Rev response elements; cPPT-centralpolypurine tract; CBA-chicken beta actin; WPRE-Woodchuck Hepatitis viruspost-transcriptional regulatory element; SIN LTR-seld inactivating LTR.

FIG. 21 shows a western blot analysis using 7E11 antibody to demonstrateNgR1 knockdown in cloned Neuroscreen cells.

FIG. 22 shows a summary of NgR expression in cloned NeuroScreen cellstransduced with LV (clone # 3G9, 1E5 1E9 1E10), LV-NgR RNAi or naïvecells. NgR and GAPDH signals on western blot results were quantified bydensitometry scanning.

FIG. 23 shows four NeuroScreen cell clones that were established withdifferent levels of NgR knockdown. NgR expression is shown as thepercentage of the NgR:GAPDH signals ratio in naïve NeuroScreen cells.

FIGS. 24A-B show the effect of ectodomain of the rat NgR1 (27-310) fusedto a rat IgG [NgR(310)ecto-Fc] and methylprednisolone (MP) onmyelin-induced inhibition of neurite outgrowth in chick dorsal rootganglia (DRGs) in vitro. (A) Dissociated embryonic day 13 chick DRGneurons were plated on phosphate-buffered saline (PBS) or myelin (400ng/well) in the presence of NgR(310)ecto-Fc or MP. (B) Quantification ofneurite outgrowth per cell (η-3) expressed as a percentage of PBScontrol±SEM (η-3). Scale bar, 200 μm. P<0/05 compared with PBS control.

FIGS. 25A-E show the effect of ecto-domain of the rat NgR1 (27-310)fused to a rat IgG [NgR(310)ecto-Fc] and methylprednisolone (MP) onfunctional recovery after spinal cord injury (SCI). (A) BBB score wasrecorded weekly for 4 weeks. (B) BBB score in MP-treated rats 2 daysafter SCI. (C) BBB scores normalized to day 2 for individual animals.(D) Frequency of consistent plantar stepping and hindlimb-forelimbcoordination, illustrating the proportion of rats in each group thatattained a score of 14 or higher 3 and 4 weeks after SCI. (E) Meanstride length in NgR(310)ecto-Fc- and MP+NgR(310)ecto-Fc-treated groupscompared with controls.

FIGS. 26A-B show the effect of ecto-domain of the rat NgR1 (27-310)fused to a rat IgG [NgR(310)ecto-Fc] and methylprednisolone (MP)treatment on axon number caudal to the spinal cord lesion.

FIG. 27 shows the effect of ecto-domain of the rat NgR1 (27-310) fusedto a rat IgG [NgR(310)ecto-Fc] and methylprednisolone (NP) treatment onthe number of biotin dextran amine (BDA)-labeled axons contacting motorneurons in the ventral horn.

DETAILED DESCRIPTION OF THE INVENTION

Definitions and General Techniques

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In case of conflict, thepresent application including the definitions will control. Also, unlessotherwise required by context, singular terms shall include pluralitiesand plural terms shall include the singular. All publications, patentsand other references mentioned herein are incorporated by reference intheir entireties for all purposes.

Although methods and materials similar or equivalent to those describedherein can be used in practice or testing of the present invention,suitable methods and materials are described below. The materials,methods and examples are illustrative only, and are not intended to belimiting. Other features and advantages of the invention will beapparent from the detailed description and from the claims.

Throughout this specification and claims, the word “comprise,” orvariations such as “comprises” or “comprising,” will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers.

In order to further define this invention, the following terms anddefinitions are herein provided.

It is to be noted that the term “a” or “an” entity, refers to one ormore of that entity; for example, “an immunoglobulin molecule,” isunderstood to represent one or more immunoglobulin molecules. As such,the terms “a” (or “an”), “one or more,” and “at least one” can be usedinterchangeably herein.

As used herein, the term “consists of,” or variations such as “consistof” or “consisting of,” as used throughout the specification and claims,indicate the inclusion of any recited integer or group of integers, butthat no additional integer or group of integers may be added to thespecified method, structure or composition.

As used herein, the term “consists essentially of,” or variations suchas “consist essentially of” or “consisting essentially of,” as usedthroughout the specification and claims, indicate the inclusion of anyrecited integer or group of integers, and the optional inclusion of anyrecited integer or group of integers that do not materially change thebasic or novel properties of the specified method, structure orcomposition.

As used herein, “antibody” means an intact immunoglobulin, or anantigen-binding fragment thereof. Antibodies of this invention can be ofany isotype or class (e.g., M, D, G, E and A) or any subclass (e.g.,G1-4, A1-2) and can have either a kappa (κ) or lambda (λ) light chain.

As used herein, “Fc” means a portion of an immunoglobulin heavy chainthat comprises one or more heavy chain constant region domains, CH1, CH2and CH3. For example, a portion of the heavy chain constant region of anantibody that is obtainable by papain digestion.

As used herein, “NogoR fusion protein” means a protein comprising asoluble Nogo receptor-1 moiety fused to a heterologous polypeptide.

As used herein, “humanized antibody” means an antibody in which at leasta portion of the non-human sequences are replaced with human sequences.Examples of how to make humanized antibodies may be found in U.S. Pat.Nos. 6,054,297, 5,886,152 and 5,877,293.

As used herein, “chimeric antibody” means an antibody that contains oneor more regions from a first antibody and one or more regions from atleast one other antibody. The first antibody and the additionalantibodies can be from the same or different species.

As used herein and in U.S. patent application 60/402,866, “Nogoreceptor,” “NogoR,” “NogoR-1,” “NgR,” “NgR-1,” “NgR1” and “NGR1” eachmeans Nogo receptor-1.

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides,” and refers to amolecule composed of monomers (amino acids) linearly linked by amidebonds (also known as peptide bonds). The term “polypeptide” refers toany chain or chains of two or more amino acids, and does not refer to aspecific length of the product. Thus, peptides, dipeptides, tripeptides,oligopeptides, “protein,” “amino acid chain,” or any other term used torefer to a chain or chains of two or more amino acids, are includedwithin the definition of “polypeptide,” and the term “polypeptide” maybe used instead of, or interchangeably with any of these terms. The term“polypeptide” is also intended to refer to the products ofpost-expression modifications of the polypeptide, including withoutlimitation glycosylation, acetylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, or modification by non-naturally occurring amino acids. Apolypeptide may be derived from a natural biological source or producedby recombinant technology, but is not necessarily translated from adesignated nucleic acid sequence. It may be generated in any manner,including by chemical synthesis.

A polypeptide of the invention may be of a size of about 3 or more, 5 ormore, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 ormore, 200 or more, 500 or more, 1,000 or more, or 2,000 or more aminoacids. Polypeptides may have a defined three-dimensional structure,although they do not necessarily have such structure. Polypeptides witha defined three-dimensional structure are referred to as folded, andpolypeptides which do not possess a defined three-dimensional structure,but rather can adopt a large number of different conformations, and arereferred to as unfolded. As used herein, the term glycoprotein refers toa protein coupled to at least one carbohydrate moiety that is attachedto the protein via an oxygen-containing or a nitrogen-containing sidechain of an amino acid residue, e.g., a serine residue or an asparagineresidue.

By an “isolated” polypeptide or a fragment, variant, or derivativethereof is intended a polypeptide that is not in its natural milieu. Noparticular level of purification is required. For example, an isolatedpolypeptide can be removed from its native or natural environment.Recombinantly produced polypeptides and proteins expressed in host cellsare considered isolated for purposed of the invention, as are native orrecombinant polypeptides which have been separated, fractionated, orpartially or substantially purified by any suitable technique.

In the present invention, a “polypeptide fragment” refers to a shortamino acid sequence of a larger polypeptide. Protein fragments may be“free-standing,” or comprised within a larger polypeptide of which thefragment forms a part of region. Representative examples of polypeptidefragments of the invention, include, for example, fragments comprisingabout 5 amino acids, about 10 amino acids, about 15 amino acids, about20 amino acids, about 30 amino acids, about 40 amino acids, about 50amino acids, about 60 amino acids, about 70 amino acids, about 80 aminoacids, about 90 amino acids, and about 100 amino acids or more inlength.

The terms “fragment,” “variant,” “derivative” and “analog” whenreferring to a polypeptide of the present invention include anypolypeptide which retains at least some biological activity.Polypeptides as described herein may include fragment, variant, orderivative molecules therein without limitation, so long as thepolypeptide still serves its function. NgR1 polypeptides and polypeptidefragments of the present invention may include proteolytic fragments,deletion fragments and in particular, fragments which more easily reachthe site of action when delivered to an animal. Polypeptide fragmentsfurther include any portion of the polypeptide which comprises anantigenic or immunogenic epitope of the native polypeptide, includinglinear as well as three-dimensional epitopes. NgR1 polypeptides andpolypeptide fragments of the present invention may comprise variantregions, including fragments as described above, and also polypeptideswith altered amino acid sequences due to amino acid substitutions,deletions, or insertions. Variants may occur naturally, such as anallelic variant. By an “allelic variant” is intended alternate forms ofa gene occupying a given locus on a chromosome of an organism Genes II,Lewin, B., ed., John Wiley & Sons, New York (1985). Non-naturallyoccurring variants may be produced using art-known mutagenesistechniques. NgR1 polypeptides and polypeptide fragments of the inventionmay comprise conservative or non-conservative amino acid substitutions,deletions or additions. NGR1 polypeptides and polypeptide fragments ofthe present invention may also include derivative molecules. Variantpolypeptides may also be referred to herein as “polypeptide analogs.” Asused herein a “derivative” of a polypeptide or a polypeptide fragmentrefers to a subject polypeptide having one or more residues chemicallyderivatized by reaction of a functional side group. Also included as“derivatives” are those peptides which contain one or more naturallyoccurring amino acid derivatives of the twenty standard amino acids. Forexample, 4-hydroxyproline may be substituted for proline;5-hydroxylysine may be substituted for lysine; 3-methylhistidine may besubstituted for histidine; homoserine may be substituted for serine; andornithine may be substituted for lysine.

As used herein the term “disulfide bond” includes the covalent bondformed between two sulfur atoms. The amino acid cysteine comprises athiol group that can form a disulfide bond or bridge with a second thiolgroup.

As used herein, “fusion protein” means a protein comprising a firstpolypeptide linearly connected, via peptide bonds, to a second,polypeptide. The first polypeptide and the second polypeptide may beidentical or different, and they may be directly connected, or connectedvia a peptide linker (see below).

The term “polynucleotide” is intended to encompass a singular nucleicacid as well as plural nucleic acids, and refers to an isolated nucleicacid molecule or construct, e.g. messenger RNA (mRNA) or plasmid DNA(pDNA). A polynucleotide can contain the nucleotide sequence of thefull-length cDNA sequence, including the untranslated 5′ and 3′sequences, the coding sequences. A polynucleotide may comprise aconventional phosphodiester bond or a non-conventional bond (e.g. anamide bond, such as found in peptide nucleic acids (PNA)). Thepolynucleotide can be composed of any polyribonucleotide orpolydeoxyribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. For example, polynucleotides can be composed of single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, the polynucleotides can be composed of triple-stranded regionscomprising RNA or DNA or both RNA and DNA. polynucleotides may alsocontain one or more modified bases or DNA or RNA backbones modified forstability or for other reasons. “Modified” bases include, for example,tritylated bases and unusual bases such as inosine. A variety ofmodifications can be made to DNA and RNA; thus, “polynucleotide”embraces chemically, enzymatically, or metabolically modified forms.

The term “nucleic acid” refer to any one or more nucleic acid segments,e.g., DNA or RNA fragments, present in a polynucleotide. By “isolated”nucleic acid or polynucleotide is intended a nucleic acid molecule, DNAor RNA, which has been removed from its native environment. For example,a recombinant polynucleotide encoding an NgR polypeptide or polypeptidefragment of the invention contained in a vector is considered isolatedfor the purposes of the present invention. Further examples of anisolated polynucleotide include recombinant polynucleotides maintainedin heterologous host cells or purified (partially or substantially)polynucleotides in solution. Isolated RNA molecules include in vivo orin vitro RNA transcripts of polynucleotides of the present invention.Isolated polynucleotides or nucleic acids according to the presentinvention further include such molecules produced synthetically. Inaddition, polynucleotide or a nucleic acid may be or may include aregulatory element such as a promoter, ribosome binding site, or atranscription terminator.

As used herein, a “coding region” is a portion of nucleic acid whichconsists of codons translated into amino acids. Although a “stop codon”(TAG, TGA, or TAA) is not translated into an amino acid, it may beconsidered to be part of a coding region, but any flanking sequences,for example promoters, ribosome binding sites, transcriptionalterminators, introns, and the like, are not part of a coding region. Twoor more coding regions of the present invention can be present in asingle polynucleotide construct, e.g., on a single vector, or inseparate polynucleotide constructs, e.g., on separate (different)vectors. Furthermore, any vector may contain a single coding region, ormay comprise two or more coding regions, e.g., a single vector mayseparately encode an immunoglobulin heavy chain variable region and animmunoglobulin light chain variable region. In addition, a vector,polynucleotide, or nucleic acid of the invention may encode heterologouscoding regions, either fused or unfused to a nucleic acid encoding anNgR polypeptide or polypeptide fragment of the present invention.Heterologous coding regions include without limitation specializedelements or motifs, such as a secretory signal peptide or a heterologousfunctional domain.

In certain embodiments, the polynucleotide or nucleic acid is DNA. Inthe case of DNA, a polynucleotide comprising a nucleic acid whichencodes a polypeptide normally may include a promoter and/or othertranscription or translation control elements operably associated withone or more coding regions. An operable association is when a codingregion for a gene product, e.g., a polypeptide, is associated with oneor more regulatory sequences in such a way as to place expression of thegene product under the influence or control of the regulatorysequence(s). Two DNA fragments (such as a polypeptide coding region anda promoter associated therewith) are “operably associated” if inductionof promoter function results in the transcription of mRNA encoding thedesired gene product and if the nature of the linkage between the twoDNA fragments does not interfere with the ability of the expressionregulatory sequences to direct the expression of the gene product orinterfere with the ability of the DNA template to be transcribed. Thus,a promoter region would be operably associated with a nucleic acidencoding a polypeptide if the promoter was capable of effectingtranscription of that nucleic acid. The promoter may be a cell-specificpromoter that directs substantial transcription of the DNA only inpredetermined cells. Other transcription control elements, besides apromoter, for example enhancers, operators, repressors, andtranscription termination signals, can be operably associated with thepolynucleotide to direct cell-specific transcription. Suitable promotersand other transcription control regions are disclosed herein.

A variety of transcription control regions are known to those skilled inthe art. These include, without limitation, transcription controlregions which function in vertebrate cells, such as, but not limited to,promoter and enhancer segments from cytomegaloviruses (the immediateearly promoter, in conjunction with intron-A), simian virus 40 (theearly promoter), and retroviruses (such as Rous sarcoma virus). Othertranscription control regions include those derived from vertebrategenes such as actin, heat shock protein, bovine growth hormone andrabbit β-globin, as well as other sequences capable of controlling geneexpression in eukaryotic cells. Additional suitable transcriptioncontrol regions include tissue-specific promoters and enhancers as wellas lympholine-inducible promoters (e.g., promoters inducible byinterferons or interleukins).

Similarly, a variety of translation control elements are known to thoseof ordinary skill in the art. These include, but are not limited toribosome binding sites, translation initiation and termination codons,and elements derived from picornaviruses (particularly an internalribosome entry site, or IRES, also referred to as a CITE sequence).

In other embodiments, a polynucleotide of the present invention is RNA,for example, in the form of messenger RNA (mRNA).

Polynucleotide and nucleic acid coding regions of the present inventionmay be associated with additional coding regions which encode secretoryor signal peptides, which direct the secretion of a polypeptide encodedby a polynucleotide of the present invention. According to the signalhypothesis, proteins secreted by mammalian cells have a signal peptideor secretory leader sequence which is cleaved from the mature proteinonce export of the growing protein chain across the rough endoplasmicreticulum has been initiated. Those of ordinary skill in the art areaware that polypeptides secreted by vertebrate cells generally have asignal peptide fused to the N-terminus of the polypeptide, which iscleaved from the complete or “full length” polypeptide to produce asecreted or “mature” form of the polypeptide. In certain embodiments,the native signal peptide, e.g., an immunoglobulin heavy chain or lightchain signal peptide is used, or a functional derivative of thatsequence that retains the ability to direct the secretion of thepolypeptide that is operably associated with it. Alternatively, aheterologous mammalian signal peptide, or a functional derivativethereof, may be used. For example, the wild-type leader sequence may besubstituted with the leader sequence of human tissue plasminogenactivator (TPA) or mouse β-glucuronidase.

As used herein the term “engineered” includes manipulation of nucleicacid or polypeptide molecules by synthetic means (e.g. by recombinanttechniques, in vitro peptide synthesis, by enzymatic or chemicalcoupling of peptides or some combination of these techniques).

As used herein, the terms “linked,” “fused” and “fusion” are usedinterchangeably. These terms refer to the joining together of two moreelements or components, by whatever means including chemical conjugationor recombinant means. An “in-frame fusion” refers to the joining of twoor more polynucleotide open reading frames (ORFs) to form a continuouslonger ORF, in a manner that maintains the correct translational readingframe of the original ORFs. Thus, a recombinant fusion protein is asingle protein containing two or more segments that correspond topolypeptides encoded by the original ORFs (which segments are notnormally so joined in nature.) Although the reading frame is thus madecontinuous throughout the fused segments, the segments may be physicallyor spatially separated by, for example, in-frame linker sequence.

A “linker” sequence is a series of one or more amino acids separatingtwo polypeptide coding regions in a fusion protein. A typical linkercomprises at least 5 amino acids. Additional linkers comprise at least10 or at least 15 amino acids. In certain embodiments, the amino acidsof a peptide linker are selected so that the linker is hydrophilic. Thelinker (Gly-Gly-Gly-Gly-Ser)₃ (G4S)₃ (SEQ ID NO:65) is a preferredlinker that is widely applicable to many antibodies as it providessufficient flexibility. Other linkers include (Gly-Gly-Gly-Gly-Ser)₂(G4S)₂ (SEQ ID NO:66), Glu Ser Gly Arg Ser Gly Gly Gly Gly Ser Gly GlyGly Gly Ser (SEQ ID NO:67), Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu SerLys Ser Thr (SEQ ID NO:68), Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu SerLys Ser Thr Gln (SEQ ID NO:69), Glu Gly Lys Ser Ser Gly Ser Gly Ser GluSer Lys Val Asp (SEQ ID NO:70), Gly Ser Thr Ser Gly Ser Gly Lys Ser SerGlu Gly Lys Gly (SEQ ID NO:71), Lys Glu Ser Gly Ser Val Ser Ser Glu GlnLeu Ala Gln Phe Arg Ser Leu Asp (SEQ ID NO:72), and Glu Ser Gly Ser ValSer Ser Glu Glu Leu Ala Phe Arg Ser Leu Asp (SEQ ID NO:73). Examples ofshorter linkers include fragments of the above linkers, and examples oflonger linkers include combinations of the linkers above, combinationsof fragments of the linkers above, and combinations of the linkers abovewith fragments of the linkers above.

In the context of polypeptides, a “linear sequence” or a “sequence” isan order of amino acids in a polypeptide in an amino to carboxylterminal direction in which residues that neighbor each other in thesequence are contiguous in the primary structure of the polypeptide.

The term “expression” as used herein refers to a process by which a geneproduces a biochemical, for example, an RNA or polypeptide. The processincludes any manifestation of the functional presence of the gene withinthe cell including, without limitation, gene knockdown as well as bothtransient expression and stable expression. It includes, withoutlimitation, transcription of the gene into messenger RNA (mRNA),transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA(siRNA) or any other RNA product, and the translation of such mRNA intopolypeptide(s), as well as any processes which regulate eithertranscription or translation. If the final desired product is abiochemical, expression includes the creation of that biochemical andany precursors. Expression of a gene produces a “gene product.” As usedherein, a gene product can be either a nucleic acid, e.g., a messengerRNA produced by transcription of a gene, or a polypeptide which istranslated from a transcript. Gene products described herein furtherinclude nucleic acids with post transcriptional modifications, e.g.,polyadenylation, or polypeptides with post translational modifications,e.g., methylation, glycosylation, the addition of lipids, associationwith other protein subunits, proteolytic cleavage, and the like.

The term “RNA interference” or “RNAi” refers to the silencing ordecreasing of gene expression by siRNAs. It is the process ofsequence-specific, post-transcriptional gene silencing in animals andplants, initiated by siRNA that is homologous in its duplex region tothe sequence of the silenced gene. The gene may be endogenous orexogenous to the organism, integrated into a chromosome or present in atransfection vector that is not integrated into the genome. Theexpression of the gene is either completely or partially inhibited. RNAimay also be considered to inhibit the function of a target RNA; thefunction of the target RNA may be complete or partial.

As used herein, the terms “treat” or “treatment” refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) an undesiredphysiological change or disorder, such as the progression of multiplesclerosis. Beneficial or desired clinical results include, but are notlimited to, alleviation of symptoms, diminishment of extent of disease,stabilized (i.e., not worsening) state of disease, delay or slowing ofdisease progression, amelioration or palliation of the disease state,and remission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment. Those in need oftreatment include those already with the condition or disorder as wellas those prone to have the condition or disorder or those in which thecondition or disorder is to be prevented.

By “subject” or “individual” or “animal” or “patient” or “mammal,” ismeant any subject, particularly a mammalian subject, for whom diagnosis,prognosis, or therapy is desired. Mammalian subjects include, but arenot limited to, humans, domestic animals, farm animals, zoo animals,sport animals, pet animals such as dogs, cats, guinea pigs, rabbits,rats, mice, horses, cattle, cows; primates such as apes, monkeys,orangutans, and chimpanzees; canids such as dogs and wolves; felids suchas cats, lions, and tigers; equids such as horses, donkeys, and zebras;food animals such as cows, pigs, and sheep; ungulates such as deer andgiraffes; rodents such as mice, rats, hamsters and guinea pigs; and soon. In certain embodiments, the mammal is a human subject.

As used herein, a “therapeutically effective amount” refers to an amounteffective, at dosages and for periods of time necessary, to achieve thedesired therapeutic result. A therapeutic result may be, e.g., lesseningof symptoms, prolonged survival, improved mobility, and the like. Atherapeutic result need not be a “cure”.

As used herein, a “prophylactically effective amount” refers to anamount effective, at dosages and for periods of time necessary, toachieve the desired prophylactic result. Typically, since a prophylacticdose is used in subjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

The invention is directed to certain NgR1 antagonists that promoteneuronal survival, neurite outgrowth and axonal regeneration of neurons,for example, CNS neurons. For example, the present invention providesNgR1 polypeptides and polypeptide fragments, antibodies and fragmentsthereof, and polynucleotides which stimulate axonal growth underconditions in which axonal growth is normally inhibited. Thus, NgR1antagonists of the invention are useful in treating injuries, diseasesor disorders that can be alleviated by promoting neuronal survival, orby the stimulation of axonal growth or regeneration in the CNS.

Exemplary CNS diseases, disorders or injuries include, but are notlimited to, multiple sclerosis (MS), progressive multifocalleukoencephalopathy (PML), encephalomyelitis (EPL), central pontinemyelolysis (CPM), adrenoleukodystrophy, Alexander's disease, PelizaeusMerzbacher disease (PMZ), Globoid cell Leucodystrophy (Krabbe's disease)and Wallerian Degeneration, optic neuritis, transverse myelitis,amylotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer'sdisease, Parkinson's disease, spinal cord injury, traumatic braininjury, post radiation injury, neurologic complications of chemotherapy,stroke, acute ischemic optic neuropathy, vitamin E deficiency, isolatedvitamin E deficiency syndrome, AR, Bassen-Kornzweig syndrome,Marchiafava-Bignami syndrome, metachromatic leukodystrophy, trigeminalneuralgia, and Bell's palsy. Among these diseases, MS is the mostwidespread, affecting approximately 2.5 million people worldwide.

Nogo Receptor-1 Polypeptides

In one aspect, the present invention relates to Nogo receptor-1polypeptides that are immunogenic. In some embodiments of the invention,the immunogenic polypeptide consists essentially of an amino acidsequence selected from the group consisting of: LDLSDNAQLRVVDPTT (rat)(SEQ ID NO: 1); LDLSDNAQLRSVDPAT (human) (SEQ ID NO: 2);AVASGPFRPFQTNQLTDEELLGLPKCCQPDAADKA (rat) (SEQ ID NO: 3);AVATGPYHPIWTGRATDEEPLGLPKCCQPDAADKA (human) (SEQ ID NO: 4); andCRLGQAGSGA (mouse) (SEQ ID NO: 5).

In some embodiments, the invention relates to Nogo receptor 1polypeptides that are bound by a monoclonal antibody that binds to Nogoreceptor-1. In some embodiments, the polypeptide is recognized by the7E11 monoclonal antibody. In some embodiments, the polypeptide isselected from the group consisting of: LDLSDNAQLR (SEQ ID NO: 27);LDLSDDAELR (SEQ ID NO: 29); LDLASDNAQLR (SEQ ID NO: 30); LDLASDDAELR(SEQ ID NO: 31); LDALSDNAQLR (SEQ ID NO: 32); LDALSDDAELR (SEQ ID NO:33); LDLSSDNAQLR (SEQ ID NO: 34); LDLSSDEAELR (SEQ ID NO: 35);DNAQLRVVDPTT (SEQ ID NO: 36); DNAQLR (SEQ ID NO: 37); ADLSDNAQLRVVDPTT(SEQ ID NO: 41); LALSDNAQLRVVDPTT (SEQ ID NO: 42); LDLSDNAALRVVDPTT (SEQID NO: 43); LDLSDNAQLHVVDPTT (SEQ ID NO: 44); and LDLSDNAQLAVVDPTT (SEQID NO: 46).

In some embodiments, the invention relates to a nucleic acid encoding apolypeptide of SEQ ID NOs: 1-5, 26-27, 29-37 and 41-45. In someembodiments of the invention, the nucleic acid molecule is linked to anexpression control sequence (e.g., pCDNA(I)).

The present invention also relates to a vector comprising a nucleic acidcoding for a polypeptide of the invention. In some embodiments of theinvention, the vector is a cloning vector. In some embodiments of theinvention, the vector is an expression vector. In some embodiments ofthe invention, the vector contains at least one selectable marker.

The present invention also relates to host cells comprising theabove-described nucleic acid or vector.

The present invention also relates to a method of producing animmunogenic polypeptide of the invention comprising the step ofculturing a host cell. In some embodiments, the host cell isprokaryotic. In some embodiments, the host cell is eukaryotic. In someembodiments, the host cell is yeast.

The present invention is also directed to certain Nogo receptor-1polypeptides and polypeptide fragments useful, e.g., for promotingneurite outgrowth, promoting neuronal survival, promoting axonalsurvival, or inhibiting signal transduction by the NgR1 signalingcomplex. Typically, the polypeptides and polypeptide fragments of theinvention act to block NgR1-mediated inhibition of neuronal survival,neurite outgrowth or axonal regeneration of central nervous system (CNS)neurons.

The human NGR1 polypeptide is shown below as SEQ ID NO:49.

Full-Length Human NgR1 (SEQ ID NO:49):MKRASAGGSRLLAWVLWLQAWQVAAPCPGACVCYNEPKVTTSCPQQGLQAVPVGIPAASQRIFLHGNRISHVPAASFRACRNTILWLHSNVLARIDAAAFTGLALLEQLDLSDNAQLRSVDPATFHGLGRLHTLHDRCGLQELGPGLFRGLAALQYLYLQDNALQALPDDTFRDLGNLTHLFLHGNRISSVPERAFRGLHSLDRLLLHQNRVAHVHPHAFRDLGRLMTLYLFANNLSALPTEALAPLRALQYLRLNDNPWVCDCRARPLWAWLQKFRGSSSEVPCSLPQRLAGRDLKRLAANDLQGCAVATGPYHPIWTGRATDEEPLGLPKCCQPDAAKASVLEPGRPASAGNALKGRVPPGDSPPGNGSGPRHINDSPFGTLPGSAEPPLTAVRPEGSEPPGFPTSGPRRRPGCSRKNRTRSHCRLGQAGSGGGGTGDSEGSGALPSL TCSLTPLGLALVLWTVLGPC

The rat NgR1 polypeptide is shown below as SEQ ID NO:50.

Full-Length Rat NgR1 (SEQ ID NO:50):MKRASSGGSRLLAWVLWLQAWRVATPCPGACVCYNEPKVTTSCPQQGLQAVPTGIPASSQRIFLHGNRISHVPAASFQSCRNLTILWLHSNALARIDAAAFTGLTLLEQLDLSDNAQLHVVDPTTFHGLGHLHTLHLDRCGLRELGPGLFRGLAALQYLYLQDNNLQALPDNTFRDLGNLTHLFLHGNRIPSVPEAHAFRGLHSLDRLLLHQNHVARVHPHAFRDLGRLMTLYLFANNLSMLPAEVLMPLRSLQYLRLNDNPWVCDCRARPLWAWLQKFRGSSSEVPCNLPQRLADRDLKRLAASDLEGCAVASGPFRPIQTSQLTDEELLSLPKCCQPDAADKASVLEPGRPASAGNALKGRVPPGDTPPGNGSGPRHINDSPFGTLPSSAEPPLTALRPGGSEPPGLPTTGPRRRPGCSRKNRTRSHCRLGQAGSGASGTGDAEGSGALPALACSLAPLGLALVLWTVLGPC

The mouse NgR1 polypeptide is shown below as SEQ ID NO:51.

Full-Length Mouse NgR1 (SEQ ID NO:51):MKRASSGGSRLLAWVLWLQAWRVATPCPGACVCYNEPKVTTSCPQQGLQAVPTGIPASSQRIFLHGNRISHVPAASGQSCRNLTILWLHSNALARIDAAAFTGLTLLEQLDLSDNAQLHVVDPTTFHGLGHLHTLHLDRCGLRELGPGLFRGLAALQYLYLQDNNLQALPDNTFRDLGNLTHLFLHGNRIPSVPEHAFRGLHSLDRLLLHQNHVARVHPHAFRDLGRLMTLYLFANNLSMLPAEVLMPLRSLQYLRLNDNPWVCDCRARPLWAWLQKFRGSSSEVPCNLPQRLADRDLKRLAASDLEGCAVASGPFRPIQTSQLTDEELLSLPKCCQPDAADKASVLEPGRPASAGNALKGRVPPGDTPPGNGSGPRHINDSPFGTLPSSAEPPLTALRPGGSEPPGLPTTGPRRRPGCSRKNRTRSHCRLGQAGSGASGTGDAEGSGALPALACSLAPLGLALVLWTVLGPC

Antibodies

The present invention further relates to an antibody or anantigen-binding fragment thereof that specifically binds a Nogoreceptor-1 polypeptide of the invention. In some embodiments theantibody or antigen-binding fragment binds a polypeptide consistingessentially of an amino acid sequence selected from the group consistingof SEQ ID NOs: 1-5, 26-27, 29-37 and 41-45. The antibody orantigen-binding fragment of the present invention may be produced invivo or in vitro. Production of the antibody or antigen-binding fragmentis discussed below.

An antibody or an antigen-binding fragment thereof of the inventioninhibits the binding of Nogo receptor-1 to a ligand (e.g., NogoA, NogoB,NogoC, MAG, OM-gp) and decreases myelin-mediated inhibition of neuriteoutgrowth and sprouting, particularly axonal growth, and attenuatesmyelin mediated growth cone collapse.

in some embodiments, the anti-Nogo receptor-1 antibody orantigen-binding fragment thereof is murine. In some embodiments, theNogo receptor-1 is from rat. In other embodiments, the Nogo receptor-1is human. In some embodiments the anti-Nogo receptor-1 antibody orantigen-binding fragment thereof is recombinant, engineered, humanizedand/or chimeric.

In some embodiments, the antibody is selected from the group consistingof: monoclonal 7E11 (ATCC® accession No. PTA4587); monoclonal 1H2 (ATCC®accession No. PTA-4584); monoclonal 2F7 (ATCC® accession No. PTA-4585);monoclonal 3G5 (ATCC® accession No. PTA-4586); and monoclonal 5B10(ATCC® accession No. PTA-4588). In some embodiments, the antibody ispolyclonal antibody 46.

Exemplary antigen-binding fragments are, Fab, Fab′, F(ab′)₂, Fv, Fd,dAb, and fragments containing complementarity determining region (CDR)fragments, single-chain antibodies (scFv), chimeric antibodies,diabodies and polypeptides that contain at least a portion of animmunoglobulin that is sufficient to confer specific antigen-binding tothe polypeptide (e.g. immunoadhesins).

As used herein, Fd means a fragment that consists of the V_(H) andC_(H1) domains; Fv means a fragment that consists of the V_(L) and V_(H)domains of a single arm of an antibody; and dAb means a fragment thatconsists of a V_(H) domain (Ward et al., Nature 341:544-546 (1989)). Asused herein, single-chain antibody (scFv) means an antibody in which aV_(L) region and a V_(H) region are paired to form a monovalentmolecules via a synthetic linker that enables them to be made as asingle protein chain (Bird et al., Science 242:423-426 (1988) and Hustonet al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)). As used herein,diabody means a bispecific antibody in which V_(H) and V_(L) domains areexpressed on a single polypeptide chain, but using a linker that is tooshort to allow for pairing between the two domains on the same chain,thereby forcing the domains to pair with complementary domains ofanother chain and creating two antigen-binding sites (see e.g.,Holliger, P., et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) andPoljak, R. J., et al., Structure 2:1121-1123 (1994)). As used herein,immunoadhesin that specifically binds an antigen of interest, means amolecule in which one or more CDRs may be incorporated, eithercovalently or noncovalently.

In some embodiments, the invention provides a subunit polypeptide of aNogo receptor-1 antibody of the invention, wherein the subunitpolypeptide is selected from the group consisting of: (a) a heavy chainor a variable region thereof; and (b) a light chain or a variable regionthereof.

In some embodiments, the invention provides a nucleic acid encoding theheavy chain or the variable region thereof, or the light chain and thevariable region thereof of a subunit polypeptide of a Nogo receptor-1antibody of the invention.

In some embodiments, the invention provides a hypervariable region (CDR)of a Nogo receptor-1 antibody of the invention or a nucleic acidencoding a CDR.

Immunization

Antibodies of the invention can be generated by immunization of asuitable host (e.g., vertebrates, including humans, mice, rats, sheep,goats, pigs, cattle, horses, reptiles, fishes, amphibians, and in eggsof birds, reptiles and fish). Such antibodies may be polyclonal ormonoclonal.

In some embodiments, the host is immunized with an immunogenic Nogoreceptor-1 polypeptide of the invention. In other embodiments, the hostis immunized with Nogo receptor-1 associated with the cell membrane ofan intact or disrupted cell and antibodies of the invention areidentified by binding to a Nogo receptor-1 polypeptide of the invention.

In some embodiments, the Nogo receptor-1 antigen is administered with anadjuvant to stimulate the immune response. Adjuvants often need to beadministered in addition to antigen in order to elicit an immuneresponse to the antigen. These adjuvants are usually insoluble orundegradable substances that promote nonspecific inflammation, withrecruitment of mononuclear phagocytes at the site of immunization.Examples of adjuvants include, but are not limited to, Freund'sadjuvant, RIBI (muramyl dipeptides), ISCOM (immunostimulating complexes)or fragments thereof.

For a review of methods for making antibodies, see e.g., Harlow andLane, Antibodies, A Laboratory Manual (1988); Yelton, D. E. et al., Ann.Rev. of Biochem. 50:657-80. (1981); and Ausubel et al., CurrentProtocols in Molecular Biology (New York: John Wiley & Sons) (1989).Determination of immunoreactivity with an immunogenic Nogo receptor-1polypeptide of the invention may be made by any of several methods wellknown in the art, including, e.g., immunoblot assay and ELISA.

Production of Antibodies and Antibody Producing Cell Lines

Monoclonal antibodies of the invention can made by standard proceduresas described, e.g., in Harlow and Lane, Antibodies, A Laboratory Manual(1988), supra.

Briefly, at an appropriate period of time the animal is sacrificed andlymph node and/or splenic B-cells are immortalized by any one of severaltechniques that are well-known in the art, including but not limited totransformation, such as with EBV or fusion with an immortalized cellline, such as myeloma cells. Thereafter, the cells are clonallyseparated and the supernatants of each clone tested for production of anantibody specific for an immunogenic Nogo receptor-1 polypeptide of theinvention. Methods of selecting, cloning and expanding hybridomas arewell known in the art. Similarly, methods for identifying the nucleotideand amino acid sequence of the immunoglobulin genes are known in theart.

Other suitable techniques for producing an antibody of the inventioninvolve in vitro exposure of lymphocytes to the Nogo receptor-1 or to animmunogenic polypeptide of the invention, or alternatively, selection oflibraries of antibodies in phage or similar vectors. See Huse et al.,Science, 246:1275-81 (1989). Antibodies useful in the present inventionmay be employed with or without modification.

Antigens (in this case Nogo receptor-1 or an immunogenic polypeptide ofthe invention) and antibodies can be labeled by joining, eithercovalently or non-covalently, a substance that provides for a detectablesignal. Various labels and conjugation techniques are known in the artand can be employed in practicing the invention. Suitable labelsinclude, but are not limited to, radionucleotides, enzymes, substrates,cofactors, inhibitors, fluorescent agents, chemiluminescent agents,magnetic particles and the like. Patents teaching the use of such labelsinclude U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;4,277,437; 4,275,149 and 4,366,241. Also, recombinant immunoglobulinsmay be produced (see U.S. Pat. No. 4,816,567).

In some embodiments of the invention, an antibody has multiple bindingspecificities, such as a bifunctional antibody prepared by any one of anumber of techniques known to those of skill in the art including theproduction of hybrid hybridomas, disulfide exchange, chemicalcross-linking, addition of peptide linkers between two monoclonalantibodies, the introduction of two sets of immunoglobulin heavy andlight chains into a particular cell line, and so forth (see below formore detailed discussion).

The antibodies of this invention may also be human monoclonalantibodies, for example those produced by immortalized human cells, bySCID-hu mice or other non-human animals capable of producing “‘human’ ”antibodies.

Phage Display Libraries

Anti-Nogo receptor-1 antibodies of this invention can be isolated byscreening a recombinant combinatorial antibody library. Exemplarycombinatorial libraries are for binding to an immunogenic Nogoreceptor-1 polypeptide of the invention, such as a scFv phage displaylibrary, prepared using V_(L) and V_(H) cDNAs prepared from mRNA derivedan animal immunized with an immunogenic Nogo receptor-1 polypeptide ofthe invention. Methodologies for preparing and screening such librariesare known in the art. There are commercially available methods andmaterials for generating phage display libraries (e.g., the PharmaciaRecombinant Phage Antibody System, catalog no. 27-9400-01; theStratagene SurfZAP™ phage display it, catalog no. 240612; and othersfrom MorphoSys). There are also other methods and reagents that can beused in generating and screening antibody display libraries (see e.g.,Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCT Publication No.WO 92/18619; Dower et al. PCT Publication No. WO 91/17271; Winter et al.PCT Publication No. WO 92/20791; Markland et al. PCT Publication No. WO92/15679; Breitling et al. PCT Publication No. WO 93/01288; McCaffertyet al. PCT Publication No. WO 92/01047; Garrard et al. PCT PublicationNo. WO 92/09690; Fuchs et al., Bio/Technology 9:1370-1372 (1991); Hay etal., Hum. Antibod. Hybridomas 3:81-85; (1992) Huse et al., Science246:1275-1281 (1989); McCafferty et al., Nature 348:552-554 (1990);Griffiths et al., EMBO J. 12:725-734 (1993); Hawkins et al., J. Mol.Biol. 226:889-896 (1992); Clackson et al., Nature 352:624-628 (1991);Gram et al., Proc. Natl. Acad. Sci. USA 89:3576-3580 (1992); Garrad etal., Bio/Technology 9:1373-1377 (1991); Hoogenboom et al., Nucl. AcidsRes. 19:4133-4137 (1991); and Barbas et al, Proc. Natl Acad. Sci. USA88:7978-7982 (1991).

Following screening and isolation of an anti-Nogo receptor-1 antibody ofthe invention from a recombinant immunoglobulin display library, thenucleic acid encoding the selected antibody can be recovered from thedisplay package (e.g., from the phage genome) and subcloned into otherexpression vectors by standard recombinant DNA techniques. If desired,the nucleic acid can be further manipulated to create other antibodyforms of the invention, as described below. To express an antibodyisolated by screening a combinatorial library, DNA encoding the antibodyheavy chain and light chain or the variable regions thereof is clonedinto a recombinant expression vector and introduced into a mammalianhost cell, as described above.

Class Switching

Anti-Nogo receptor-1 antibodies of the invention can be of any isotype.An antibody of any desired isotype can be produced by class switching.For class switching, nucleic acids encoding V_(L) or V_(H), that do notinclude any nucleotide sequences encoding C_(L) or C_(H), are isolatedusing methods well known in the art. The nucleic acids encoding V_(L) orV_(H) are then operatively linked to a nucleotide sequence encoding aC_(L) or C_(H) from a desired class of immunoglobulin molecule. This maybe achieved using a vector or nucleic acid that comprises a C_(L) orC_(H) chain, as described above. For example, an anti-Nogo receptor-1antibody of the invention that was originally IgM may be class switchedto an IgG. Further, the class switching may be used to convert one IgGsubclass to another, e.g., from IgG1 to IgG2.

Mutated Antibodies

In other embodiments, antibodies or antigen-binding fragments of theinvention may be mutated in the variable domains of the heavy and/orlight chains to alter a binding property of the antibody. For example, amutation may be made in one or more of the CDR regions to increase ordecrease the K_(d) of the antibody for Nogo receptor-1, to increase ordecrease K_(off) or to alter the binding specificity of the antibody.Techniques in site-directed mutagenesis are well known in the art. Seee.g., Sambrook et al., Molecular Cloning—A Laboratory Manual, ColdSpring Harbor Laboratory Press (1989) and Ausubel et al, CurrentProtocols in Molecular Biology (New York: John Wiley & Sons) (1989). Ina preferred embodiment, mutations are made at an amino acid residue thatis known to be changed compared to germline in a variable region of ananti-Nogo receptor-1 antibody of the invention. In some embodiments,mutations are made at one or more amino acid residues that are known tobe changed compared to the germline in a variable region of an anti-Nogoreceptor-1 antibody of the invention. In another embodiment, a nucleicacid encoding an antibody heavy chain or light chain variable region ismutated in one or more of the framework regions. A mutation may be madein a framework region or constant domain to increase the half-life. Amutation in a framework region or constant domain also may be made toalter the immunogenicity of the antibody, to provide a site for covalentor non-covalent binding to another molecule, or to alter such propertiesas complement fixation. Mutations may be made in each of the frameworkregions, the constant domain and the variable regions in a singlemutated antibody. Alternatively, mutations may be made in only one ofthe framework regions, the variable regions or the constant domain in asingle mutated antibody.

Fusion Antibodies and Immunoadhesins

In another embodiment, a fusion antibody or immunoadhesin may be madewhich comprises all or a portion of an anti-Nogo receptor-1 antibody ofthe invention linked to another polypeptide. In some embodiments, onlythe variable region of the anti-Nogo receptor-1 antibody is linked tothe polypeptide. In other embodiments, the V_(H) domain of an anti-Nogoreceptor-1 antibody of this invention is linked to a first polypeptide,while the V_(L) domain of the antibody is linked to a second polypeptidethat associates with the first polypeptide in a manner that permits theV_(H) and V_(L) domains to interact with one another to form an antibodybinding site. In other embodiments, the V_(H) domain is separated fromthe V_(L) domain by a linker that permits the V_(H) and V_(L) domains tointeract with one another (see below under Single Chain Antibodies). TheV_(H)-linker-V_(L) antibody is then linked to a polypeptide of interest.The fusion antibody is useful to directing a polypeptide to a cell ortissue that expresses a Nogo receptor-1 ligand. The polypeptide ofinterest may be a therapeutic agent, such as a toxin, or may be adiagnostic agent, such as an enzyme that may be easily visualized, suchas horseradish peroxidase. In addition, fusion antibodies can be createdin which two (or more) single-chain antibodies are linked to oneanother. This is useful if one wants to create a divalent or polyvalentantibody on a single polypeptide chain, or if one wants to create abispecific antibody.

Single Chain Antibodies

The present invention includes a single chain antibody (scFv) that bindsa Nogo receptor-1 polypeptide of the invention. To produce the ScFv,V_(H)- and V_(L)-encoding DNA is operatively linked to DNA encoding aflexible linker, e.g., encoding the amino acid sequence (Gly₄-Ser)₃ (SEQID NO: 10), such that the V_(H) and V_(L) sequences can be expressed asa contiguous single-chain protein, with the V_(L) and V_(H) regionsjoined by the flexible linker (see, e.g., Bird et al., Science242:423-426 (1988); Huston et al, Proc. Natl. Acad. Sci. USA85:5879-5883 (1988); McCafferty et al., Nature 348:552-554 (1990)). Thesingle chain antibody may be monovalent, if only a single V_(H) andV_(L) are used, bivalent, if two V_(H) and V_(L) are used, orpolyvalent, if more than two V_(H) and V_(L) are used.

Chimeric Antibodies

The present invention further includes a bispecific antibody orantigen-binding fragment thereof in which one specificity is for a Nogoreceptor-1 polypeptide of the invention. In one embodiment, a chimericantibody can be generated that specifically binds to a Nogo receptor-1polypeptide of the invention through one binding domain and to a secondmolecule through a second binding domain. The chimeric antibody can beproduced through recombinant molecular biological techniques, or may bephysically conjugated together. In addition, a single chain antibodycontaining more than one V_(H) and V_(L) may be generated that bindsspecifically to a polypeptide of the invention and to another moleculethat is associated with attenuating myelin mediated growth cone collapseand inhibition of neurite outgrowth and sprouting. Such bispecificantibodies can be generated using techniques that are well known forexample, Fanger et al., Immunol Methods 4: 72-81 (1994) and Wright andHarris, supra. and in connection with (iii) see e.g. Traunecker et al.Int. J. Cancer (Suppl.) 7: 51-52 (1992).

In some embodiments, the chimeric antibodies are prepared using one ormore of the variable regions from an antibody of the invention. Inanother embodiment, the chimeric antibody is prepared using one or moreCDR regions from said antibody.

Derivatized and Labeled Antibodies

An antibody or an antigen-binding fragment of the invention can bederivatized or linked to another molecule (e.g., another peptide orprotein). In general, the antibody or antigen-binding fragment isderivatized such that binding to a polypeptide of the invention is notaffected adversely by the derivatization or labeling. For example, anantibody or antibody portion of the invention can be functionally linked(by chemical coupling, genetic fusion, noncovalent association orotherwise) to one or more other molecular entities, such as anotherantibody (e.g. a bispecific antibody or a diabody), a detection agent, acytotoxic agent, a pharmaceutical agent, and/or a protein or peptidethat can mediate association of the antibody or antigen-binding fragmentwith another molecule (such as a streptavidin core region or apolyhistidine tag).

In some embodiments, a derivatized antibody is produced by crosslinkingtwo or more antibodies (of the same type or of different types, e.g., tocreate bispecific antibodies). Suitable crosslinkers include those thatare heterobifunctional, having two distinctly reactive groups separatedby an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimideester) or homobifunctional (e.g., disuccinimidyl suberate). Such linkersare available from Pierce Chemical Company, Rockford, Ill.

In some embodiments, the derivatized antibody is a labeled antibody.Exemplary, detection agents with which an antibody or antibody portionof the invention may be derivatized are fluorescent compounds, includingfluorescein, fluorescein isothiocyanate, rhodamine,5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, lanthanidephosphors and the like. An antibody also may be labeled with enzymesthat are useful for detection, such as horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase andthe like. In embodiments that are labeled with a detectable enzyme, theantibody is detected by adding additional reagents that the enzyme usesto produce a detectable reaction product. For example, horseradishperoxidase with hydrogen peroxide and diaminobenzidine. An antibody alsomay be labeled with biotin, and detected through indirect measurement ofavidin or streptavidin binding. An antibody may also be labeled with apredetermined polypeptide epitopes recognized by a secondary reporter(e.g., leucine zipper pair sequences, binding sites for secondaryantibodies, metal binding domains, epitope tags).

An anti-Nogo receptor-1 antibody or an antigen-fragment thereof also maybe labeled with a radio-labeled amino acid. The radiolabel may be usedfor both diagnostic and therapeutic purposes. The radio-labeledanti-Nogo receptor-1 antibody may be used diagnostically, for example,for determining Nogo receptor-1 levels in a subject. Further, theradio-labeled anti-Nogo receptor-1 antibody may be used therapeuticallyfor treating spinal cord injury. Examples of labels for polypeptidesinclude, but are not limited to, the following radioisotopes orradionucleotides—³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I.

An anti-Nogo receptor-1 antibody or an antigen-fragment thereof may alsobe derivatized with a chemical group such as polyethylene glycol (PEG),a methyl or ethyl group, or a carbohydrate group. These groups may beuseful to improve the biological characteristics of the antibody, e.g.,to increase serum half-life or to increase tissue binding.

Characterization of Anti-Nogo Receptor-1 Antibodies

Class and Subclass of Anti-Nogo Receptor-1 Antibodies

The class and subclass of anti-Nogo receptor-1 antibodies may bedetermined by any method known in the art. In general, the class andsubclass of an antibody may be determined using antibodies that arespecific for a particular class and subclass of antibody. Suchantibodies are available commercially. The class and subclass can bedetermined by ELISA, Western Blot as well as other techniques.Alternatively, the class and subclass may be determined by sequencingall or a portion of the constant domains of the heavy and/or lightchains of the antibodies, comparing their amino acid sequences to theknown amino acid sequences of various class and subclasses ofimmunoglobulins, and determining the class and subclass of theantibodies.

Binding Affinity of Anti-Nogo Receptor-1 Antibody to Nogo Receptor-1

The binding affinity and dissociation rate of an anti-Nogo receptor-1antibody of the invention to a Nogo receptor-1 polypeptide of theinvention may be determined by any method known in the art. For example,the binding affinity can be measured by competitive ELISAs, RIAs,BIAcore or KinExA technology. The dissociation rate also can be measuredby BIAcore or KinExA technology. The binding affinity and dissociationrate are measured by surface plasmon resonance using, e.g. a BIAcore.The K_(d) of 7E11 and 1H2 were determined to be 1×10⁻⁷ M and 2×10⁻⁸ M,respectively.

Inhibition of Nogo Receptor-1 Activity by Anti-Nogo Receptor-1 Antibody

In some embodiments, an anti-Nogo receptor-1 antibody or anantigen-binding fragment of the invention thereof inhibits the bindingof Nogo receptor-1 to a ligand. The IC₅₀ of such inhibition can bemeasured by any method known in the art, e.g., by ELISA, RIA, orFunctional Antagonism. In some embodiments, the IC₅₀ is between 0.1 and500 nM. In some embodiments, the IC₅₀ is between 10 and 400 nM. In yetother embodiments, the antibody or portion thereof has an IC₅₀ ofbetween 60 nM and 400 nM. The IC₅₀ of 7E11 and 1H2 were determined to be400 nM and 60 nM, respectively, in a binding assay. See also Table 3,infra.

In some embodiments, the present invention also include NgR1-specificantibodies or antigen-binding fragments, variants, or derivatives whichare antagonists of NgR1 activity. For example, the binding of certainNgR1 antibodies to NgR1 blocks NgR1-mediated inhibition of neuronalsurvival, neurite outgrowth or axonal regeneration of central nervoussystem (CNS) neurons.

In other embodiments, the present invention includes an antibody, orantigen-binding fragment, variant, or derivative thereof whichspecifically or preferentially binds to at least one epitope of NgR1,where the epitope comprises, consists essentially of, or consists of atleast about four to five amino acids of SEQ ID NO:49, at least seven, atleast nine, or between at least about 15 to about 30 amino acids of SEQID NO:49. The amino acids of a given epitope of SEQ ID NO:49 asdescribed may be, but need not be contiguous or linear. In certainembodiments, the at least one epitope of NgR1 comprises, consistsessentially of, or consists of a non-linear epitope formed by theextracellular domain of NgR1 as expressed on the surface of a cell or asa soluble fragment, e.g., fused to an IgG Fc region. Thus, in certainembodiments the at least one epitope of NgR1 comprises, consistsessentially of, or consists of at least 4, at least 5, at least 6, atleast 7, at least 8, at least 9, at least 10, at least 15, at least 20,at least 25, between about 15 to about 30, or at least 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100contiguous or non-contiguous amino acids of SEQ ID NO:49, wherenon-contiguous amino acids form an epitope through protein folding.

In other embodiments, the present invention includes an antibody, orantigen-binding fragment, variant, or derivative thereof whichspecifically or preferentially binds to at least one epitope of NgR1,where the epitope comprises, consists essentially of, or consists of, inaddition to one, two, three, four, five, six or more contiguous ornon-contiguous amino acids of SEQ ID NO:49 as described above, and anadditional moiety which modifies the protein, e.g., a carbohydratemoiety may be included such that the NgR1 antibody binds with higheraffinity to modified target protein than it does to an unmodifiedversion of the protein. Alternatively, the NgR1 antibody does not bindthe unmodified version of the target protein at all.

In certain embodiments, an antibody, or antigen-binding fragment,variant, or derivative thereof of the invention binds specifically to atleast one epitope of NGR1 or fragment or variant described above, i.e.,binds to such an epitope more readily than it would bind to anunrelated, or random epitope; binds preferentially to at least oneepitope of NgR1 or fragment or variant described above, i.e., binds tosuch an epitope more readily than it would bind to a related, similar,homologous, or analogous epitope; competitively inhibits binding of areference antibody which itself binds specifically or preferentially toa certain epitope of NgR1 or fragment or variant described above; orbinds to at least one epitope of NGR1 or fragment or variant describedabove with an affinity characterized by a dissociation constant KD ofless than about 5×10⁻² M, about 10⁻² M, about 5×10⁻³ M, about 10⁻³ M,about 5×10⁻⁴ M, about 10⁻⁴ M, about 5×10⁻⁵ M, about 10⁻⁵ M, about 5×10⁻⁶M, about 10⁻⁶ M, about 5×10⁻⁷ M, about 10⁻⁷ M, about 5×10⁻⁸ M, about10⁻⁸ M, about 5×10⁻⁹ M, about 10⁻⁹ M, about 5×10⁻¹⁰ M, about 10⁻¹⁰ M,about 5×10⁻¹¹ M, about 10⁻¹¹ M, about 5×10⁻¹² M, about 10⁻¹² M, about5×10⁻¹³ M, about 10⁻¹³ M, about 5×10⁻¹⁴ M, about 10⁻¹⁴ M, about 5×10⁻¹⁵M, or about 10⁻¹⁵ M. In a particular aspect, the antibody or fragmentthereof preferentially binds to a human NgR1 polypeptide or fragmentthereof, relative to a murine NgR1 polypeptide or fragment thereof.

As used in the context of antibody binding dissociation constants, theterm “about” allows for the degree of variation inherent in the methodsutilized for measuring antibody affinity. For example, depending on thelevel of precision of the instrumentation used, standard error based onthe number of samples measured, and rounding error, the term “about 10⁻²M” might include, for example, from 0.05 M to 0.005 M.

In specific embodiments, an antibody, or antigen-binding fragment,variant, or derivative thereof of the invention binds NGR1 polypeptidesor fragments or variants thereof with an off rate (k(off)) of less thanor equal to 5×10⁻² sec⁻¹, 10⁻² sec⁻¹, 5×10⁻³ sec⁻¹ or 10⁻³ sec⁻¹.Alternatively, an antibody, or antigen-binding fragment, variant, orderivative thereof of the invention binds NgR1 polypeptides or fragmentsor variants thereof with an off rate (k(off)) of less than or equal to5×10⁻⁴ sec⁻¹, 10⁻⁴ sec⁻¹, 5×10⁻⁵ sec⁻¹, or 10⁻⁵ sec⁻¹ 5×10⁻⁶ sec⁻¹, 10⁻⁶sec⁻¹, 5×10⁻⁷ sec⁻¹ or 10⁻⁷ sec⁻¹.

In other embodiments, an antibody, or antigen-binding fragment, variant,or derivative thereof of the invention binds NgR1 polypeptides orfragments or variants thereof with an on rate (k(on)) of greater than orequal to 10³ M⁻¹ sec⁻¹, 5×10³ M⁻¹ sec⁻¹, 10⁴ M⁻¹ sec⁻¹ or 5×10⁴ M⁻¹sec⁻¹. Alternatively, an antibody, or antigen-binding fragment, variant,or derivative thereof of the invention binds NgR1 polypeptides orfragments or variants thereof with an on rate (k(on)) greater than orequal to 10⁵ M⁻¹ sec⁻¹, 5×10⁵ M⁻¹ sec⁻¹, 10⁶ M⁻¹ sec⁻¹, or 5×10⁶ M⁻¹sec⁻¹ or 10⁷ M⁻¹ sec⁻¹.

In one embodiment, a NgR1 antagonist for use in the methods of theinvention is an antibody molecule, or immunospecific fragment thereof.Unless it is specifically noted, as used herein a “fragment thereof” inreference to an antibody refers to an immunospecific fragment, i.e., anantigen-specific fragment. In one embodiment, an antibody of theinvention is a bispecific binding molecule, binding polypeptide, orantibody, e.g., a bispecific antibody, minibody, domain deletedantibody, or fusion protein having binding specificity for more than oneepitope, e.g. more than one antigen or more than one epitope on the sameantigen. In one embodiment, a bispecific antibody has at least onebinding domain specific for at least one epitope on NgR1. A bispecificantibody may be a tetravalent antibody that has two target bindingdomains specific for an epitope of NgR1 and two target binding domainsspecific for a second target. Thus, a tetravalent bispecific antibodymay be bivalent for each specificity.

In certain embodiments of the present invention comprise administrationof an NgR1 antagonist antibody, or immunospecific fragment thereof, inwhich at least a fraction of one or more of the constant region domainshas been deleted or otherwise altered so as to provide desiredbiochemical characteristics such as reduced effector functions, theability to non-covalently dimerize, increased ability to localize at thesite of a tumor, reduced serum half-life, or increased serum half-lifewhen compared with a whole, unaltered antibody of approximately the sameimmunogenicity. For example, certain antibodies for use in the treatmentmethods described herein are domain deleted antibodies which comprise apolypeptide chain similar to an immunoglobulin heavy chain, but whichlack at least a portion of one or more heavy chain domains. Forinstance, in certain antibodies, one entire domain of the constantregion of the modified antibody will be deleted, for example, all orpart of the CH2 domain will be deleted.

In certain NgR1 antagonist antibodies or immunospecific fragmentsthereof for use in the therapeutic methods described herein, the Fcportion may be mutated to alter, e.g., increase, decrease or modulateeffector function using techniques known in the art. For example, thedeletion or inactivation (through point mutations or other means) of aconstant region domain may reduce or alter Fc receptor binding of thecirculating modified antibody thereby increasing tumor localization. Inother cases it may be that constant region modifications consistent withthe instant invention moderate complement binding and thus reduce theserum half life and nonspecific association of a conjugated cytotoxin.Yet other modifications of the constant region may be used to modifydisulfide linkages or oligosaccharide moieties that allow for enhancedlocalization due to increased antigen specificity or antibodyflexibility. The resulting physiological profile, bioavailability andother biochemical effects of the modifications, such as tumorlocalization, biodistribution and serum half-life, may easily bemeasured and quantified using well know immunological techniques withoutundue experimentation.

Modified forms of antibodies or immunospecific fragments thereof for usein the diagnostic and therapeutic methods disclosed herein can be madefrom whole precursor or parent antibodies using techniques known in theart. Exemplary techniques are discussed in more detail herein.

In certain embodiments both the variable and constant regions of NgR1antagonist antibodies or immunospecific fragments thereof for use in thetreatment methods disclosed herein are fully human. Fully humanantibodies can be made using techniques that are known in the art and asdescribed herein. For example, fully human antibodies against a specificantigen can be prepared by administering the antigen to a transgenicanimal which has been modified to produce such antibodies in response toantigenic challenge, but whose endogenous loci have been disabled.Exemplary techniques that can be used to make such antibodies aredescribed in U.S. Pat. Nos. 6,150,584; 6,458,592; 6,420,140. Othertechniques are known in the art. Fully human anti bodies can likewise beproduced by various display technologies, e.g., phage display or otherviral display systems, as described in more detail elsewhere herein.

NgR1 antagonist antibodies or immunospecific fragments thereof for usein the diagnostic and treatment methods disclosed herein can be made ormanufactured using techniques that are known in the art. In certainembodiments, antibody molecules or fragments thereof are “recombinantlyproduced,” i.e., are produced using recombinant DNA technology.Exemplary techniques for making antibody molecules or fragments thereofare discussed in more detail elsewhere herein.

NgR1 antagonist antibodies or immunospecific fragments thereof for usein the treatment methods disclosed herein include derivatives that aremodified, e.g., by the covalent attachment of any type of molecule tothe antibody such that covalent attachment does not prevent the antibodyfrom specifically binding to its cognate epitope. For example, but notby way of limitation, the antibody derivatives include antibodies thathave been modified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

In preferred embodiments, an NgR1 antagonist antibody or immunospecificfragment thereof for use in the treatment methods disclosed herein willnot elicit a deleterious immune response in the animal to be treated,e.g., in a human. In one embodiment, NgR1 antagonist antibodies orimmunospecific fragments thereof for use in the treatment methodsdisclosed herein may be modified to reduce their immunogenicity usingart-recognized techniques. For example, antibodies can be humanized,primatized, deimmunized, or chimeric antibodies can be made. These typesof antibodies are derived from a non-human antibody, typically a murineor primate antibody, that retains or substantially retains theantigen-binding properties of the parent antibody, but which is lessimmunogenic in humans. This may be achieved by various methods,including (a) grafting the entire non-human variable domains onto humanconstant regions to generate chimeric antibodies; (b) grafting at leasta part of one or more of the non-human complementarity determiningregions (CDRs) into a human framework and constant regions with orwithout retention of critical framework residues; or (c) transplantingthe entire non-human variable domains, but “cloaking” them with ahuman-like section by replacement of surface residues. Such methods aredisclosed in Morrison et al., Proc. Natl. Acad. Sci. 81:6851-6855(1984); Morrison et al., Adv. Immunol. 44:65-92 (1988); Verhoeyen etal., Science 239:1534-1536 (1988); Padlan, Molec. Immun. 28:489-498(1991); Padlan, Molec. Immun. 31:169-217 (1994), and U.S. Pat. Nos.5,585,089, 5,693,761, 5,693,762, and 6,190,370, all of which are herebyincorporated by reference in their entirety.

De-immunization can also be used to decrease the immunogenicity of anantibody. As used herein, the term “de-immunization” includes alterationof an antibody to modify T cell epitopes (see, e.g., WO9852976A1,WO0034317A2). For example, V_(H) and V_(L) sequences from the startingantibody are analyzed and a human T cell epitope “map” from each, Vregion showing the location of epitopes in relation tocomplementarity-determining regions (CDRs) and other key residues withinthe sequence. Individual T cell epitopes from the T cell epitope map areanalyzed in order to identify alternative amino acid substitutions witha low risk of altering activity of the final antibody. A range ofalternative V_(H) and V_(L) sequences are designed comprisingcombinations of amino acid substitutions and these sequences aresubsequently incorporated into a range of binding polypeptides, e.g.,NGR1 antagonist antibodies or immunospecific fragments thereof for usein the diagnostic and treatment methods disclosed herein, which are thentested for function. Typically, between 12 and 24 variant antibodies aregenerated and tested. Complete heavy and light chain genes comprisingmodified V and human C regions are then cloned into expression vectorsand the subsequent plasmids introduced into cell lines for theproduction of whole antibody. The antibodies are then compared inappropriate biochemical and biological assays, and the optimal variantis identified.

NGR1 antagonist antibodies or fragments thereof for use in the methodsof the present invention may be generated by any suitable method knownin the art. Polyclonal antibodies can be produced by various procedureswell known in the art. For example, a NgR1 immunospecific fragment canbe administered to various host animals including, but not limited to,rabbits, mice, rats, etc. to induce the production of sera containingpolyclonal antibodies specific for the antigen. Various adjuvants may beused to increase the immunological response, depending on the hostspecies, and include but are not limited to, Freund's (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanins, dinitrophenol, andpotentially useful human adjuvants such as BCG (bacille Calmette-Guerin)and Corynebacterium parvum. Such adjuvants are also well known in theart.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed.(1988); Hammerling et al., in: Monoclonal Antibodies and T-CellHybridomas Elsevier, N.Y., 563-681 (1981) (said references incorporatedby reference in their entireties). The term “monoclonal antibody” asused herein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced. Thus, the term“monoclonal antibody” is not limited to antibodies produced throughhybridoma technology. Monoclonal antibodies can be prepared using a widevariety of techniques known in the art including the use of hybridomaand recombinant and phage display technology.

Using art recognized protocols, in one example, antibodies are raised inmammals by multiple subcutaneous or intraperitoneal injections of therelevant antigen (e.g., purified NgR1 antigens or cells or cellularextracts comprising such antigens) and an adjuvant. This immunizationtypically elicits an immune response that comprises production ofantigen-reactive antibodies from activated splenocytes or lymphocytes.While the resulting antibodies may be harvested from the serum of theanimal to provide polyclonal preparations, it is often desirable toisolate individual lymphocytes from the spleen, lymph nodes orperipheral blood to provide homogenous preparations of monoclonalantibodies (MAbs). Preferably, the lymphocytes are obtained from thespleen.

In this well known process (Kohler et al., Nature 256:495 (1975)) therelatively short-lived, or mortal, lymphocytes from a mammal which hasbeen injected with antigen are fused with an immortal tumor cell line(e.g. a myeloma cell line), thus, producing hybrid cells or “hybridomas”which are both immortal and capable of producing the genetically codedantibody of the B cell. The resulting hybrids are segregated into singlegenetic strains by selection, dilution, and regrowth with eachindividual strain comprising specific genes for the formation of asingle antibody. They produce antibodies which are homogeneous against adesired antigen and, in reference to their pure genetic parentage, aretermed “monoclonal.”

Hybridoma cells thus prepared are seeded and grown in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, parental myeloma cells. Those skilledin the art will appreciate that reagents, cell lines and media for theformation, selection and growth of hybridomas are commercially availablefrom a number of sources and standardized protocols are wellestablished. Generally, culture medium in which the hybridoma cells aregrowing is assayed for production of monoclonal antibodies against thedesired antigen. Preferably, the binding specificity of the monoclonalantibodies produced by hybridoma cells is determined by in vitro assayssuch as immunoprecipitation, radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA). After hybridoma cells are identified thatproduce antibodies of the desired specificity, affinity and/or activity,the clones may be subcloned by limiting dilution procedures and grown bystandard methods (Goding, Monoclonal Antibodies: Principles andPractice, Academic Press, pp 59-103 (1986)). It will further beappreciated that the monoclonal antibodies secreted by the subclones maybe separated from culture medium, ascites fluid or serum by conventionalpurification procedures such as, for example, protein-A, hydroxylapatitechromatography, gel electrophoresis, dialysis or affinitychromatography.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)2 fragments may be producedby proteolytic cleavage of immunoglobulin molecules, using enzymes suchas papain (to produce Fab fragments) or pepsin (to produce F(ab′)2fragments). F(ab′)2 fragments contain the variable region, the lightchain constant region and the CH1 domain of the heavy chain.

Those skilled in the art will also appreciate that DNA encodingantibodies or antibody fragments (e.g., antigen binding sites) may alsobe derived from antibody phage libraries. In a particular, such phagecan be utilized to display antigen-binding domains expressed from arepertoire or combinatorial antibody library (e.g., human or murine).Phage expressing an antigen binding domain that binds the antigen ofinterest can be selected or identified with antigen, e.g., using labeledantigen or antigen bound or captured to a solid surface or bead. Phageused in these methods are typically filamentous phage including fd andM13 binding domains expressed from phage with Fab, Fv or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Exemplary methods are set forth, forexample, in EP 368 684 B1; U.S. Pat. No. 5,969,108, Hoogenboom, H. R.and Chames, Immunol. Today 21:371 (2000); Nagy et al. Nat. Med. 8:801(2002); Huie et al., Proc. Natl. Acad. Sci. USA 98:2682 (2001); Lui etal., J. Mol. Biol. 315:1063 (2002), each of which is incorporated hereinby reference. Several publications (e.g., Marks et al., Bio/Technology10:779-783 (1992)) have described the production of high affinity humanantibodies by chain shuffling, as well as combinatorial infection and invivo recombination as a strategy for constructing large phage libraries.In another embodiment, Ribosomal display can be used to replacebacteriophage as the display platform (see, e.g., Hanes et al., Nat.Biotechnol. 18:1287 (2000); Wilson et al., Proc. Natl. Acad. Sci. USA98:3750 (2001); or Irving et al., J. Immunol. Methods 248:31 (2001)). Inyet another embodiment, cell surface libraries can be screened forantibodies Coder et al., Proc. Natl. Acad. Sci. USA 97:10701 (2000);Daugherty et al., J. Immunol. Methods 243:211 (2000)). Such proceduresprovide alternatives to traditional hybridoma techniques for theisolation and subsequent cloning of monoclonal antibodies.

In phage display methods, functional antibody domains are displayed onthe surface of phage particles which carry the polynucleotide sequencesencoding them. In particular, DNA sequences encoding V_(H) and V_(L)regions are amplified from animal cDNA libraries (e.g., human or murinecDNA libraries of lymphoid tissues) or synthetic cDNA libraries. Incertain embodiments, the DNA encoding the V_(H) and V_(L) regions arejoined together by an scFv linker by PCR and cloned into a phagemidvector (e.g., p CANTAB 6 or pComb 3 HSS). The vector is electroporatedin E. coli and the E. coli is infected with helper phage. Phage used inthese methods are typically filamentous phage including fd and M13 andthe V_(H) or V_(L) regions are usually recombinantly fused to either thephage gene III or gene VIII. Phage expressing an antigen binding domainthat binds to an antigen of interest (i.e., a NgR1 polypeptide or afragment thereof) can be selected or identified with antigen, e.g.,using labeled antigen or antigen bound or captured to a solid surface orbead.

Additional examples of phage display methods that can be used to makethe antibodies include those disclosed in Brinkman et al., J. Immunol.Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186(1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persicet al., Gene 187:9-18 (1997); Burton et al., Advances in Immunology57:191-280 (1994); PCT Application No. PCT/GB91/01134; PCT publicationsWO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108;each of which is incorporated herein by reference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria. For example, techniques to recombinantly produce Fab, Fab′ andF(ab′)2 fragments can also be employed using methods known in the artsuch as those disclosed in PCT publication WO 92/22324; Mullinax et al.,BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34(1995); and Better et al., Science 240:1041-1043 (1988) (said referencesincorporated by reference in their entireties).

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu etal., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040(1988). For some uses, including in vivo use of antibodies in humans andin vitro detection assays, it may be preferable to use chimeric,humanized, or human antibodies. A chimeric antibody is a molecule inwhich different portions of the antibody are derived from differentanimal species, such as antibodies having a variable region derived froma murine monoclonal antibody and a human immunoglobulin constant region.Methods for producing chimeric antibodies are known in the art. See,e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214(1986); Gillies et al., J. Immunol. Methods 125:191-202 (1989); U.S.Pat. Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporatedherein by reference in their entireties. Humanized antibodies areantibody molecules from non-human species antibody that binds thedesired antigen having one or more complementarity determining regions(CDRs) from the non-human species and framework regions from a humanimmunoglobulin molecule. Often, framework residues in the humanframework regions will be substituted with the corresponding residuefrom the CDR donor antibody to alter, preferably improve, antigenbinding. These framework substitutions are identified by methods wellknown in the art, e.g., by modeling of the interactions of the CDR andframework residues to identify framework residues important for antigenbinding and sequence comparison to identify unusual framework residuesat particular positions. (See, e.g., Queen et al., U.S. Pat. No.5,585,089; Riechmann et al., Nature 332:323 (1988), which areincorporated herein by reference in their entireties.) Antibodies can behumanized using a variety of techniques known in the art including, forexample, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S.Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing(EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska. et al, PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332).

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735, and WO 91/10741; each of which is incorporatedherein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring that express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g. all or aportion of a desired target polypeptide. Monoclonal antibodies directedagainst the antigen can be obtained from the immunized, transgenic miceusing conventional hybridoma technology. The human immunoglobulintransgenes harbored by the transgenic mice rearrange during B-celldifferentiation, and subsequently undergo class switching and somaticmutation. Thus, using such a technique, it is possible to producetherapeutically useful IgG, IgA, IgM and IgE antibodies. For an overviewof this technology for producing human antibodies, see Lonberg andHuszar, Int. Rev. Immunol. 13:65-93 (1995). For a detailed discussion ofthis technology for producing human antibodies and human monoclonalantibodies and protocols for producing such antibodies, see, e.g., PCTpublications WO 98/24893; WO 96/34096; WO 96/33735; U.S. Pat. Nos.5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806;5,814,318; and 5,939,598, which are incorporated by reference herein intheir entirety. In addition, companies such as Abgenix, Inc. (Freemont,Calif.) and GenPharm (San Jose, Calif.) can be engaged to provide humanantibodies directed against a selected antigen using technology similarto that described above.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/Technology 12:899-903(1988)). See also, U.S. Pat. No. 5,565,332.

In another embodiment, DNA encoding desired monoclonal antibodies may bereadily isolated and sequenced using conventional procedures (e.g., byusing oligonucleotide probes that are capable of binding specifically togenes encoding the heavy and light chains of murine antibodies). Theisolated and subcloned hybridoma cells serve as a preferred source ofsuch DNA. Once isolated, the DNA may be placed into expression vectors,which are then transfected into prokaryotic or eukaryotic host cellssuch as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO)cells or myeloma cells that do not otherwise produce immunoglobulins.More particularly, the isolated DNA (which may be synthetic as describedherein) may be used to clone constant and variable region sequences forthe manufacture antibodies as described in Newman et al., U.S. Pat. No.5,658,570, filed Jan. 25, 1995, which is incorporated by referenceherein. Essentially, this entails extraction of RNA from the selectedcells, conversion to cDNA, and amplification by PCR using Ig specificprimers. Suitable primers for this purpose are also described in U.S.Pat. No. 5,658,570. As will be discussed in more detail below,transformed cells expressing the desired antibody may be grown up inrelatively large quantities to provide clinical and commercial suppliesof the immunoglobulin.

In a specific embodiment, the amino acid sequence of the heavy and/orlight chain variable domains may be inspected to identify the sequencesof the complementarity determining regions (CDRs) by methods that arewell know in the art, e.g., by comparison to known amino acid sequencesof other heavy and light chain variable regions to determine the regionsof sequence hypervariability. Using routine recombinant DNA techniques,one or more of the CDRs may be inserted within framework regions, e.g.,into human framework regions to humanize a non-human antibody. Theframework regions may be naturally occurring or consensus frameworkregions, and preferably human framework regions (see, e.g., Chothia etal., J. Mol. Biol. 278:457-479 (1998) for a listing of human frameworkregions). Preferably, the polynucleotide generated by the combination ofthe framework regions and CDRs encodes an antibody that specificallybinds to at least one epitope of a desired polypeptide, e.g. NgR1.Preferably, one or more amino acid substitutions may be made within theframework regions, and, preferably, the amino acid substitutions improvebinding of the antibody to its antigen. Additionally, such methods maybe used to make amino acid substitutions or deletions of one or morevariable region cysteine residues participating in an intrachaindisulfide bond to generate antibody molecules lacking one or moreintrachain disulfide bonds. Other alterations to the polynucleotide areencompassed by the present invention and within the skill of the art.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al, Proc. Natl. Acad. Sci. 81:851-855 (1984);Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature314:452-454 (1985)) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Asused herein, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine monoclonal antibody and a humanimmunoglobulin constant region, e.g., humanized antibodies.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,694,778; Bird, Science 242:423-442 (1988);Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Wardet al., Nature 334:544-554 (1989)) can be adapted to produce singlechain antibodies. Single chain antibodies are formed by linking theheavy and light chain fragments of the Fv region via an amino acidbridge, resulting in a single chain antibody. Techniques for theassembly of functional Fv fragments in E coli may also be used (Skerraet al., Science 242:1038-1041 (1988)).

NgR1 antagonist antibodies may also be human or substantially humanantibodies generated in transgenic animals (e.g., mice) that areincapable of endogenous immunoglobulin production (see e.g., U.S. Pat.Nos. 6,075,181, 5,939,598, 5,591,669 and 5,589,369 each of which isincorporated herein by reference). For example, it has been describedthat the homozygous deletion of the antibody heavy-chain joining regionin chimeric and germ-line mutant mice results in complete inhibition ofendogenous antibody production. Transfer of a human immunoglobulin genearray to such germ line mutant mice will result in the production ofhuman antibodies upon antigen challenge. Another preferred means ofgenerating human antibodies using SCID mice is disclosed in U.S. Pat.No. 5,811,524 which is incorporated herein by reference. It will beappreciated that the genetic material associated with these humanantibodies may also be isolated and manipulated as described herein.

Yet another highly efficient means for generating recombinant antibodiesis disclosed by Newman, Biotechnology 10: 1455-1460 (1992).Specifically, this technique results in the generation of primatizedantibodies that contain monkey variable domains and human constantsequences. This reference is incorporated by reference in its entiretyherein. Moreover, this technique is also described in commonly assignedU.S. Pat. Nos. 5,658,570, 5,693,780 and 5,756,096 each of which isincorporated herein by reference.

In another embodiment, lymphocytes can be selected by micromanipulationand the variable genes isolated. For example, peripheral bloodmononuclear cells can be isolated from an immunized mammal and culturedfor about 7 days in vitro. The cultures can be screened for specificIgGs that meet the screening criteria. Cells from positive wells can beisolated. Individual Ig-producing B cells can be isolated by FACS or byidentifying them in a complement-mediated hemolytic plaque assay.Ig-producing B cells can be micromanipulated into a tube and the V_(H)and V_(L) genes can be amplified using, e.g., RT-PCR. The V_(H) andV_(L) genes can be cloned into an antibody expression vector andtransfected into cells (e.g., eukaryotic or prokaryotic cells) forexpression.

Alternatively, antibody-producing cell lines may be selected andcultured using techniques well known to the skilled artisan. Suchtechniques are described in a variety of laboratory manuals and primarypublications. In this respect, techniques suitable for use in theinvention as described below are described in Current Protocols inImmunology, Coligan et al., Eds., Green Publishing Associates andWiley-Interscience, John Wiley and Sons, New York (1991) which is hereinincorporated by reference in its entirety, including supplements.

Antibodies for use in the therapeutic methods disclosed herein can beproduced by any method known in the art for the synthesis of antibodies,in particular, by chemical synthesis or preferably, by recombinantexpression techniques as described herein.

It will further be appreciated that the scope of this invention furtherencompasses all alleles, variants and mutations of antigen binding DNAsequences.

As is well known, RNA may be isolated from the original hybridoma cellsor from other transformed cells by standard techniques, such asguanidinium isothiocyanate extraction and precipitation followed bycentrifugation or chromatography. Where desirable, mRNA may be isolatedfrom total RNA by standard techniques such as chromatography on oligo dTcellulose. Suitable techniques are familiar in the art.

In one embodiment, cDNAs that encode the light and the heavy chains ofthe antibody may be made, either simultaneously or separately, usingreverse transcriptase and DNA polymerase in accordance with well knownmethods. PCR may be initiated by consensus constant region primers or bymore specific primers based on the published heavy and light chain DNAand amino acid sequences. As discussed above, PCR also may be used toisolate DNA clones encoding the antibody light and heavy chains. In thiscase the libraries may be screened by consensus primers or largerhomologous probes, such as mouse constant region probes.

DNA, typically plasmid DNA, may be isolated from the cells usingtechniques known in the art, restriction mapped and sequenced inaccordance with standard, well known techniques set forth in detail,e.g., in the foregoing references relating to recombinant DNAtechniques. Of course, the DNA may be synthetic according to the presentinvention at any point during the isolation process or subsequentanalysis.

Recombinant expression of an antibody, or fragment, derivative or analogthereof, e.g. a heavy or light chain of an antibody which is an NgR1antagonist, requires construction of an expression vector containing apolynucleotide that encodes the antibody. Once a polynucleotide encodingan antibody molecule or a heavy or light chain of an antibody, orportion thereof (preferably containing the heavy or light chain variabledomain), of the invention has been obtained, the vector for theproduction of the antibody molecule may be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody encoding nucleotide sequence are described herein. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. The invention,thus, provides replicable vectors comprising a nucleotide sequenceencoding an antibody molecule of the invention, or a heavy or lightchain thereof, or a heavy or light chain variable domain, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g., PCTPublication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.5,122,464) and the variable domain of the antibody may be cloned intosuch a vector for expression of the entire heavy or light chain.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody for use in the methods describedherein. Thus, the invention includes host cells containing apolynucleotide encoding an antibody of the invention, or a heavy orlight chain thereof, operably linked to a heterologous promoter. Inpreferred embodiments for the expression of double-chained antibodies,vectors encoding both the heavy and light chains may be co-expressed inthe host cell for expression of the entire immunoglobulin molecule, asdetailed below.

A variety of host-expression vector systems may be utilized to expressantibody molecules for use in the methods described herein. Suchhost-expression systems represent vehicles by which the coding sequencesof interest may be produced and subsequently purified, but alsorepresent cells which may, when transformed or transfected with theappropriate nucleotide coding sequences, express an antibody molecule ofthe invention in situ. These include but are not limited tomicroorganisms such as bacteria (e.g., E. coli, B. subtilis) transformedwith recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expressionvectors containing antibody coding sequences; yeast (e.g.,Saccharomyces, Pichia) transformed with recombinant yeast expressionvectors containing antibody coding sequences; insect cell systemsinfected with recombinant virus expression vectors (e.g., baculovirus)containing antibody coding sequences; plant cell systems infected withrecombinant virus expression vectors (e.g., cauliflower mosaic virus,CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmidexpression vectors (e.g., Ti plasmid) containing antibody codingsequences; or mammalian cell systems (e.g., COS, CHO, BLK, 293, 3T3cells) harboring recombinant expression constructs containing promotersderived from the genome of mammalian cells (e.g., metallothioneinpromoter) or from mammalian viruses (e.g., the adenovirus late promoter;the vaccinia virus 7.5K promoter). Preferably, bacterial cells such asEscherichia coli, and more preferably, eukaryotic cells, especially forthe expression of whole recombinant antibody molecule, are used for theexpression of a recombinant antibody molecule. For example, mammaliancells such as Chinese hamster ovary cells (CHO), in conjunction with avector such as the major intermediate early gene promoter element fromhuman cytomegalovirus is an effective expression system for antibodies(Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2(1990)).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791(1983)), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lacZ coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, NucleicAcids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.24:5503-5509 (1989)); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding to amatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is typically used as a vector to express foreign genes. Thevirus grows in Spodoptera frugiperda cells. The antibody coding sequencemay be cloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g. the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts. (e.g., see Logan &Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specificinitiation signals may also be required for efficient translation ofinserted antibody coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al., Methodsin Enzymol. 153:51-544 (1987)).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, HeLa, COS, MDCK,293, 3T3, WI38, and in particular, breast cancer cell lines such as, forexample, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary glandcell line such as, for example, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which stably express theantibody molecule.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223(1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817 1980) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Proc. Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc.Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G418Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991);Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan,Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem.62:191-217 (1993); TIB TECH 11(5):155-215 (May, 1993); and hygro, whichconfers resistance to hygromycin (Santerre et al., Gene 30:147 (1984).Methods commonly known in the art of recombinant DNA technology whichcan be used are described in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transferand Expression, A Laboratory Manual, Stockton Press, NY (1990); and inChapters 12 and 13, Dracopoli et al. (eds), Current Protocols in HumanGenetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol.Biol. 150:1 (1981), which are incorporated by reference herein in theirentireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Academic Press, New York, Vol. 3.(1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257(1983)).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes both heavy and light chainpolypeptides. In such situations, the light chain is advantageouslyplaced before the heavy chain to avoid an excess of toxic free heavychain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad. Sci.USA 77:2197 (1980)). The coding sequences for the heavy and light chainsmay comprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been recombinantlyexpressed, it may be purified by any method known in the art forpurification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins.Alternatively, a preferred method for increasing the affinity ofantibodies of the invention is disclosed in US 2002 0123057 A1.

In one embodiment, a binding molecule or antigen binding molecule foruse in the methods of the invention comprises a synthetic constantregion wherein one or more domains are partially or entirely deleted(“domain-deleted antibodies”). In certain embodiments compatiblemodified antibodies will comprise domain deleted constructs or variantswherein the entire CH2 domain has been removed (ΔC_(H)2 constructs). Forother embodiments a short connecting peptide may be substituted for thedeleted domain to provide flexibility and freedom of movement for thevariable region. Those skilled in the art will appreciate that suchconstructs are particularly preferred due to the regulatory propertiesof the CH2 domain on the catabolic rate of the antibody.

In certain embodiments, modified antibodies for use in the methodsdisclosed herein are minibodies. Minibodies can be made using methodsdescribed in the art (see, e.g., U.S. Pat. No. 5,837,821 or WO94/09817A1).

In another embodiment, modified antibodies for use in the methodsdisclosed herein are CH2 domain deleted antibodies which are known inthe art. Domain deleted constructs can be derived using a vector (e.g.,from Biogen IDEC Incorporated) encoding an IgG1 human constant domain(see, e.g., WO 02/060955A2 and WO 02/096948A2). This exemplary vectorwas engineered to delete the CH2 domain and provide a synthetic vectorexpressing a domain deleted IgG1 constant region.

In one embodiment, a NgR1 antagonist antibody or fragment thereof foruse in the treatment methods disclosed herein comprises animmunoglobulin heavy chain having deletion or substitution of a few oreven a single amino acid as long as it permits association between themonomeric subunits. For example, the mutation of a single amino acid inselected areas of the CH2 domain may be enough to substantially reduceFc binding and thereby increase tumor localization. Similarly, it may bedesirable to simply delete that part of one or more constant regiondomains that control the effector function (e.g. complement binding) tobe modulated. Such partial deletions of the constant regions may improveselected characteristics of the antibody (serum half-life) while leavingother desirable functions associated with the subject constant regiondomain intact. Moreover, as alluded to above, the constant regions ofthe disclosed antibodies may be synthetic through the mutation orsubstitution of one or more amino acids that enhances the profile of theresulting construct. In this respect it may be possible to disrupt theactivity provided by a conserved binding site (e.g., Fc binding) whilesubstantially maintaining the configuration and immunogenic profile ofthe modified antibody. Yet other embodiments comprise the addition ofone or more amino acids to the constant region to enhance desirablecharacteristics such as effector function or provide for more cytotoxinor carbohydrate attachment. In such embodiments it may be desirable toinsert or replicate specific sequences derived from selected constantregion domains.

The present invention also provides the use of antibodies that comprise,consist essentially of, or consist of, variants (including derivatives)of antibody molecules (e.g., the VH regions and/or VL regions) describedherein, which antibodies or fragments thereof immunospecifically bind toa NGR1 polypeptide. Standard techniques known to those of skill in theart can be used to introduce mutations in the nucleotide sequenceencoding a binding molecule, including, but not limited to,site-directed mutagenesis and PCR-mediated mutagenesis which result inamino acid substitutions. Preferably, the variants (includingderivatives) encode less than 50 amino acid substitutions, less than 40amino acid substitutions, less than 30 amino acid substitutions, lessthan 25 amino acid substitutions, less than 20 amino acid substitutions,less than 15 amino acid substitutions, less than 10 amino acidsubstitutions, less than 5 amino acid substitutions, less than 4 aminoacid substitutions, less than 3 amino acid substitutions, or less than 2amino acid substitutions relative to the reference VH region, VHCDR1,VHCDR2, VHCDR3, VL region, VLCDR1, VLCDR2, or VLCDR3. A “conservativeamino acid substitution” is one in which the amino acid residue isreplaced with an amino acid residue having a side chain with a similarcharge. Families of amino acid residues having side chains with similarcharges have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Alternatively, mutations can be introduced randomly alongall or part of the coding sequence, such as by saturation mutagenesis,and the resultant mutants can be screened for biological activity toidentify mutants that retain activity.

For example, it is possible to introduce mutations only in frameworkregions or only in CDR regions of an antibody molecule. Introducedmutations may be silent or neutral missense mutations, i.e., have no, orlittle, effect on an antibody's ability to bind antigen. These types ofmutations may be useful to optimize codon usage, or improve ahybridoma's antibody production. Alternatively, non-neutral missensemutations may alter an antibody's ability to bind antigen. The locationof most silent and neutral missense mutations is likely to be in theframework regions, while the location of most non-neutral missensemutations is likely to be in CDR, though this is not an absoluterequirement. One of skill in the art would be able to design and testmutant molecules with desired properties such as no alteration inantigen binding activity or alteration in binding activity (e.g.,improvements in antigen binding activity or change in antibodyspecificity). Following mutagenesis, the encoded protein may routinelybe expressed and the functional and/or biological activity of theencoded protein can be determined using techniques described herein orby routinely modifying techniques known in the art.

In sum, one of skill in the art, provided with the teachings of thisinvention, has available a variety of methods which may be used to alterthe biological properties of the antibodies of this invention includingmethods which would increase or decrease the stability or half-life,immunogenicity, toxicity, affinity or yield of a given antibodymolecule, or to alter it in any other way that may render it moresuitable for a particular application.

Compositions comprising, and uses of, the antibodies of the presentinvention are described below.

Soluble Nogo Receptor-1 Polypeptides

Protein

Full-length Nogo receptor-1 consists of a signal sequence, a N-terminusregion (NT), eight leucine rich repeats (LRR), a LRRCT region (a leucinerich repeat domain C-terminal of the eight leucine rich repeats), aC-terminus region (CT) and a GPI anchor (see FIG. 1).

The NgR domain designations used herein are defined as follows:

TABLE 1 Example NgR domains hNgR (SEQ rNgR (SEQ ID mNgR (SEQ ID DomainID: 49) NO: 50) NO: 51) Signal Seq.  1-26  1-26  1-26 LRRNT 27-56 27-5627-56 LRR1 57-81 57-81 57-81 LRR2  82-105  82-105  82-105 LRR3 106-130106-130 106-130 LRR4 131-154 131-154 131-154 LRR5 155-178 155-178155-178 LRR6 179-202 179-202 179-202 LRR7 203-226 203-226 203-226 LRR8227-250 227-250 227-250 LRRCT 260-309 260-309 260-309 CTS (CT Signaling)310-445 310-445 310-445 GPI 446-473 446-473 446-473

Some embodiments of the invention provide a soluble Nogo receptor-1polypeptide. Soluble Nogo receptor-1 polypeptides of the inventioncomprise an NT domain; 8 LRRs and an LRRCT domain and lack a signalsequence and a functional GPI anchor (i.e., no GPI anchor or a GPIanchor that lacks the ability to efficiently associate to a cellmembrane).

In some embodiments, a soluble Nogo receptor-1 polypeptide comprises aheterologous LRR. In some embodiments a soluble Nogo receptor-1polypeptide comprises 2, 3, 4, 5, 6, 7, or 8 heterologous LRRs. Aheterologous LRR means an LRR obtained from a protein other than Nogoreceptor-1. Exemplary proteins from which a heterologous LRR can beobtained are toll-like receptor (TLR1.2); T-cell activation leucinerepeat rich protein; deceorin; OM-gp; insulin-like growth factor bindingprotein acidic labile subunit slit and robo; and toll-like receptor 4.

In some embodiments, the invention provides a soluble Nogo receptor-1polypeptide of 319 amino acids (soluble Nogo receptor-1 344,sNogoR1-344, or sNogoR344) (residues 26-344 of SEQ ID NOs: 6 and 8 orresidues 27-344 of SEQ ID NO: 8). In some embodiments, the inventionprovides a soluble Nogo receptor-1 polypeptide of 285 amino acids(soluble Nogo receptor-1 310, sNogoR1-310, or sNogoR310) (residues26-310 of SEQ ID NOs: 7 and 9 or residues 27-310 of SEQ ID NO: 9). SeeFIG. 1.

TABLE 1 Sequences of Human and Rat Nogo receptor-1 Polypeptides SEQ IDNO: 6 MKRASAGGSRLLAWVLWLQAWQVAAPCPGACVCYNEP (human 1-344)KVTTSCPQQGLQAVPVGIPAASQRIFLHGNRISHVPAASFRACRNLTILWLHSNVLARIDAAAFTGLALLEQLDLSDNAQLRSVDPATFHGLGRLHTLHLDRCGLQELGPGLFRGLAALQYLYLQDNALQALPDDTFRDLGNLTHLFLHGNRISSVPERAFRGLHSLDRLLLHQNRVAHVHPHAFRDLGRLMTLYLFANNLSALPTEALAPLRALQYLRLNDNPWVCDCRARPLWAWLQKFRGSSSEVPCSLPQRLAGRDLKRLAANDLQGCAVATGPYHPIWTGRATDEEPLGLP KCCQPDAADKA SEQ ID NO: 7MKRASAGGSRLLAWVLWLQAWQVAAPCPGACVCYNEP (human 1-310)KVTTSCPQQGLQAVPVGIPAASQRIFLHGNRISHVPAASFRACRNLTILWLHSNVLARIDAAAFTGLALLEQLDLSDNAQLRSVDPATFHGLGRLHTLHLDRCGLQELGPGLFRGLAALQYLYLQDNALQALPDDTFRDLGNLTHLFLHGNRISSVPERAFRGLHSLDRLLLHQNRVAHVHPHAFRDLGRLMTLYLFANNLSALPTEALAPLRALQYLRLNDNPWVCDCRARPLWAWLQKFRGSSSEVPCSLPQRLAGR DLKRLAANDLQGCA SEQ ID NO: 8MKRASAGGSRLPTWVLWLQAWRVATPCPGACVCYNEP (rat 1-344)KVTTSRPQQGLQAVPAGIPAASQRIFLHGNRISYVPAASFQSCRNLTILWLHSNALAGIDAAAFTGLTLLEQLDLSDNAQLRVVDPTTFRGLGHLHTLHLDRCGLQELGPGLFRGLAALQYLYLQDNNLQALPDNTFRDLGNLTHLFLHGNRIPSVPEHAFRGLHSLDRLLLHQNHVARVHPHAFRDLGRLMTLYLFANNLSMLPAEVLVPLRSLQYLRLNDNPWVCDCRARPLWAWLQKFRGSSSGVPSNLPQRLAGRDLKRLATSDLEGCAVASGPFRPFQTNQLTDEELLGLP KCCQPDAADKA SEQ ID NO: 9MKRASSGGSRLPTWVLWLQAWRVATPCPGACVCYNEP (rat 1-310)KVTTSRPQQGLQAVPAGIPASSQRIFLHGNRISYVPAASFQSCRNLTILWLHSNALAGIDAAAFTGLTLLEQLDLSDNAQLRVVDPTTFRGLGHLHTLHLDRCGLQELGPGLFRGLAALQYLYLQDNNLQALPDNTFRDLGNLTHLFLHGNRIPSVPEHAFRGLHSLDRLLLHQNHVARVHPHAFRDLGRLMTLYLFANNLSMLPAEVLVPLRSLQYLRLNDNPWVCDCRARPLWAWLQKFRGSSSGVPSNLPQRLAGR DLKRLATSDLEGCA SEQ ID NO: 58MKRASAGGSRLLAWVLWLQAWQVAAPCPGACVCYNEP (human 1-310KVTTSCPQQGLQAVPVGIPAASQRIFLHGNRISHVPA with alaASFRACRNLTILWLHSNVLARIDAAAFTGLALLEQLD substitutionsLSDNAQLRSVDPATFHGLGRLHTLHLDRCGLQELGPG at amino acidLFRGLAALQYLYLQDNALQALPDDTFRDLGNLTHLFL positions 266HGNRISSVPERAFRGLHSLDRLLLHQNRVAHVHPHAF and 309)RDLGRLMTLYLFANNLSALPTEALAPLRALQYLRLNDNPWVCDARARPLWAWLQKFRGSSSEVPCSLPQRLAGR DLKRLAANDLQGAA SEQ ID NO: 59MKRASSGGSRLPTWVLWLQAWRVATPCPGACVCYNEP (rat 1-310KVTTSRPQQGLQAVPAGIPASSQRIFLHGNRISYVPA with alaASFQSCRNLTILWLHSNALAGIDAAAFTGLTLLEQLD substitutionsLSDNAQLRVVDPTTFRGLGHLHTLHLDRCGLQELGPG at amino acidLFRGLAALQYLYLQDNNLQALPDNTFRDLGNLTHLFL positions 266HGNRIPSVPEHAFRGLHSLDRLLLHQNHVARVHPHAF and 309)RDLGRLMTLYLFANNLSMLPAEVLVPLRSLQYLRLNDNPWVCDARARPLWAWLQKFRGSSSGVPSNLPQRLAGR DLKRLATSDLEGAA SEQ ID NO: 64MKRASAGGSRLLAWVLWLQAWQVAAPCPGACVCYNEP (human 1-344KVTTSCPQQGLQAVPVGIPAASQRIFLHGNRISHVPA with alaASFRACRNLTILWLHSNVLARIDAAAFTGLALLEQLD substitutionsLSDNAQLRSVDPATFHGLGRLHTLHLDRCGLQELGPG at amino acidLFRGLAALQYLYLQDNALQALPDDTFRDLGNLTHLFL positions 266HGNRISSVPERAFRGLHSLDRLLLHQNRVAHVHPHAF and 309)RDLGRLMTLYLFANNLSALPTEALAPLRALQYLRLNDNPWVCDARARPLWAWLQKFRGSSSEVPCSLPQRLAGRDLKRLAANDLQGAAVATGPYHPIWTGRATDEEPLGLP KCCQPDAADKA

In some embodiments of the invention, the soluble Nogo receptor-1polypeptides of the invention are used to inhibit the binding of aligand to Nogo receptor-1 and act as an antagonist of Nogo receptor-1ligands. In some embodiments of the invention, the soluble Nogoreceptor-1 polypeptides of the invention are used to decrease inhibitionof neurite outgrowth and sprouting in a neuron, such as axonal growthand to inhibit myelin mediated growth cone collapse in a neuron. In someembodiments, the neuron is a CNS neuron.

sNogoR310 and sNogoR344, surprisingly, block the binding of NogoA,NogoB, NogoC, MAG and OM-gp to Nogo receptor-1.

In another embodiment, the present invention provides an isolatedpolypeptide fragment of 60 residues or less, comprising an amino acidsequence identical to a reference amino acid sequence, except for up toone, two, three, four, ten, or twenty individual amino acidsubstitutions, wherein said reference amino acid sequence is selectedfrom the group consisting of: (a) amino acids x to 344 of SEQ ID NO:49,(b) amino acids 309 to y of SEQ ID NO:49, and (c) amino acids x to y ofSEQ ID NO:49, wherein x is any integer from 305 to 326, and y is anyinteger from 328 to 350; and wherein said polypeptide fragment inhibitsNogo-receptor-mediated neurite outgrowth inhibition. In someembodiments, the invention provides an isolated polypeptide fragment of60 residues or less, comprising an amino acid sequence identical to areference amino acid sequence, except for up to one, two, three, four,ten or twenty individual amino acid substitutions, wherein saidreference amino acid sequence is selected from the group consisting of:(a) amino acids x′ to 344 of SEQ ID NO:49, (b) amino acids 309 to y′ ofSEQ ID NO:49, and (c) amino acids x′ to y′ of SEQ ID NO:49, where x′isany integer from 300 to 326, and y′ is any integer from 328 to 360, andwherein said polypeptide fragment inhibits Nogo-receptor-mediatedneurite outgrowth inhibition.

By “an NgR1 reference amino acid sequence,” or “reference amino acidsequence” is meant the specified sequence without the introduction ofany amino acid substitutions. As one of ordinary skill in the art wouldunderstand, if there are no substitutions, the “isolated polypeptide” ofthe invention comprises an amino acid sequence which is identical to thereference amino acid sequence.

In some embodiments, the invention provides an isolated polypeptidefragment of 60 residues or less, comprising an amino acid sequenceidentical to a reference amino acid sequence, except for up to one, two,or three individual amino acid substitutions, wherein said referenceamino acid sequence is selected from the group consisting of: aminoacids 309 to 335 of SEQ ID NO:49; amino acids 309 to 344 of SEQ IDNO:49; amino acids 310 to 335 of SEQ ID NO:49; amino acids 310 to 344 ofSEQ ID NO:49; amino acids 309 to 350 of SEQ ID NO:49; amino acids 300 to344 of SEQ ID NO:49; and amino acids 315 to 344 of SEQ ID NO:49.

In one embodiment, the invention provides an isolated polypeptidefragment of 60 residues or less, comprising an amino acid sequenceidentical to a reference amino acid sequence, except for up to threeindividual amino acid substitutions, wherein said reference amino acidsequence is amino acids 309 to 344 of SEQ ID NO:49.

In one embodiment, the invention provides an isolated polypeptidefragment of 60 residues or less, comprising an amino acid sequenceidentical to a reference amino acid sequence, except for up to threeindividual amino acid substitutions, wherein said reference amino acidsequence is amino acids 309 to 335 of SEQ ID NO:49.

In one embodiment, the invention provides an isolated polypeptidecomprising: (a) an amino acid sequence identical to a reference aminoacid sequence except that at least one cysteine residue of saidreference amino acid sequence is substituted with a different aminoacid, wherein said reference amino acid sequence is selected from thegroup consisting of: (i) amino acids a to 445 of SEQ ID NO:49, (ii)amino acids 27 to b of SEQ ID NO:49, and (iii) amino acids a to b of SEQID NO:49, wherein a is any integer from 25 to 35, and b is any integerfrom 300 to 450; and (b) a heterologous polypeptide; wherein saidpolypeptide inhibits nogo-receptor-mediated neurite outgrowthinhibition.

Exemplary amino acid substitutions for polypeptide fragments accordingto this embodiment include substitutions of individual cysteine residuesin the polypeptides of the invention with different amino acids. Anydifferent amino acid may be substituted for a cysteine in thepolypeptides of the invention. Which different amino acid is useddepends on a number of criteria, for example, the effect of thesubstitution on the conformation of the polypeptide fragment, the chargeof the polypeptide fragment, or the hydrophilicity of the polypeptidefragment. Amino acid substitutions for the amino acids of thepolypeptides of the invention and the reference amino acid sequence caninclude amino acids with basic side chains (e.g., lysine, arginine,histidine), acidic side chains (e.g. aspartic acid, glutamic acid),uncharged polar side chains (e.g., glycine, asparagine, glutamine,serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Typical amino acids to substitutefor cysteines in the reference amino acid include alanine, serine,threonine, in particular, alanine. Making such substitutions throughengineering of a polynucleotide encoding the polypeptide fragment iswell within the routine expertise of one of ordinary skill in the art.

In another embodiment, the present invention provides an isolatedpolypeptide of the invention wherein at least one cysteine residue issubstituted with a different amino acid. Cysteine residues that cansubstituted in human NgR1 include C27, C31, C33, C43, C80, C140, C264,C266, C287, C309, C335, C336, C419, C429, C455 and C473. Cysteineresidues that can substituted in rat NgR1 include C27, C31, C33, C80,C140, C264, C266, C287, C309, C335, C336, C419, C429, C455 and C473.Cysteine residues that can substituted in mouse NgR1 include C27, C31,C33, C43, C80, C140, C264, C266, C287, C309, C335, C336, C419, C429,C455 and C473.

The present invention further provides an isolated polypeptide fragmentof 40 residues or less, where the polypeptide fragment comprises anamino acid sequence identical to amino acids 309 to 344 of SEQ ID NO:49,except that: C309 is substituted, C335 is substituted, C336 issubstituted, C309 and C335 are substituted, C309 and C336 aresubstituted, C335 and C336 are substituted, or C309, C335, and C336 aresubstituted.

The cysteine residues in the polypeptides of the invention may besubstituted with any heterologous amino acid. In certain embodiments,the cysteine is substituted with a small uncharged amino acid which isleast likely to alter the three dimensional conformation of thepolypeptide, e.g., alanine, serine, threonine, preferably alanine.

In some embodiments, the soluble Nogo receptor-1 polypeptide of theinvention is a component of a fusion protein that further comprises aheterologous polypeptide. In some embodiments, the heterologouspolypeptide is an immunoglobulin constant domain. In some embodiments,the immunoglobulin constant domain is a heavy chain constant domain. Insome embodiments, the heterologous polypeptide is an Fc fragment. Insome embodiments the Fc is joined to the C-terminal end of the solubleNogo receptor-1 polypeptide of the invention. In some embodiments thefusion Nogo receptor-1 protein is a dimer. The invention furtherencompasses variants, analogs, or derivatives of polypeptide fragmentsas described above.

In some embodiments, the invention provides an isolated polypeptidecomprising: (a) an amino acid sequence identical to a reference aminoacid sequence, except for up to twenty individual amino acidsubstitutions, wherein said reference amino acid sequence is selectedfrom the group consisting of: (i) amino acids a to 445 of SEQ ID NO:49,(ii) amino acids 27 to b of SEQ ID NO:49, and (iii) amino acids a to bof SEQ ID NO:49, wherein a is any integer from 1 to 35, and b is anyinteger from 300 to 450; and (b) a heterologous polypeptide; whereinsaid polypeptide inhibits nogo-receptor-mediated neurite outgrowthinhibition. In some embodiments, the isolated polypeptide is amino acids1 to 310 of SEQ ID NO:49, wherein R269 and A310 are substituted with adifferent amino acid. Exemplary amino acids that can be substituted inthe polypeptide include amino acids with basic side chains (e.g.,lysine, arginine, histidine), acidic side chains (e.g. aspartic acid,glutamic acid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, typtophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). In one embodiment, the differentamino acid is tryptophan.

An exemplary soluble NgR-Fc fusion protein is human NgR1(319)-Fc whichcomprises Fc joined to the C-terminal end of amino acids 1 to 319 of SEQID NO:49.

Exemplary soluble NgR-Fc fusion proteins with cysteine substitutions areAla-Ala-human(h)NgR1(310)-Fc which comprises Fc joined to the C-terminalend of a soluble polypeptide with the amino acid sequence of SEQ IDNO:58, Ala-Ala-rat(r)NgR1(310)-Fc which comprises Fc joined to theC-terminal end of a soluble polypeptide with the amino acid sequence ofSEQ ID NO:59 and Ala-Ala-human(h)NgR1 (344)-Fc which comprises Fc joinedto the C-terminal end of a soluble polypeptide with the amino acidsequence of SEQ ID NO:64.

In the present invention, a polypeptide can be composed of amino acidsjoined to each other by peptide bonds or modified peptide bonds, i.e.,peptide isosteres, and may contain amino acids other than the 20gene-encoded amino acids (e.g., non-naturally occurring amino acids).The polypeptides of the present invention may be modified by eithernatural processes, such as posttranslational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in the polypeptide, including thepeptide backbone, the amino acid side-chains and the amino or carboxyltermini. It will be appreciated that the same type of modification maybe present in the same or varying degrees at several sites in a givenpolypeptide. Also, a given polypeptide may contain many types ofmodifications. Polypeptides may be branched, for example, as a result ofubiquitination, and they may be cyclic, with or without branching.Cyclic, branched, and branched cyclic polypeptides may result fromnatural processes or may be made by synthetic methods. Modificationsinclude, but are not limited to, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, transfer-RNA mediated additionof amino acids to proteins such as arginylation, and ubiquitination.(See, for instance, Proteins—Structure And Molecular Properties, 2ndEd., T. E. Creighton, W.H. Freeman and Company, New York (1993);Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol182:626-646 (1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992).).

Polypeptides described herein may be cyclic. Cyclization of thepolypeptides reduces the conformational freedom of linear peptides andresults in a more structurally constrained molecule. Many methods ofpeptide cyclization are known in the art. For example, “backbone tobackbone” cyclization by the formation of an amide bond between theN-terminal and the C-terminal amino acid residues of the peptide. The“backbone to backbone” cyclization method includes the formation ofdisulfide bridges between two α-thio amino acid residues (e.g. cysteine,homocysteine). Certain peptides of the present invention includemodifications on the N- and C-terminus of the peptide to form a cyclicpolypeptide. Such modifications include, but are not limited, tocysteine residues, acetylated cysteine residues, cysteine residues witha NH2 moiety and biotin. Other methods of peptide cyclization aredescribed in Li & Roller, Curr. Top. Med. Chem. 3:325-341 (2002) andU.S. Patent Publication No. U.S. 2005-0260626 A1, which are incorporatedby reference herein in their entirety.

In methods of the present invention, an NgR1 polypeptide or polypeptidefragment of the invention can be administered directly as a preformedpolypeptide, or indirectly through a nucleic acid vector. In someembodiments of the invention, an NGR1 polypeptide or polypeptidefragment of the invention is administered in a treatment method thatincludes: (1) transforming or transfecting an implantable host cell witha nucleic acid, e.g., a vector, that expresses an NgR1 polypeptide orpolypeptide fragment of the invention; and (2) implanting thetransformed host cell into a mammal, at the site of a disease, disorderor injury. For example, the transformed host cell can be implanted atthe site of a chronic lesion of MS. In some embodiments of theinvention, the implantable host cell is removed from a mammal,temporarily cultured, transformed or transfected with an isolatednucleic acid encoding an NgR1 polypeptide or polypeptide fragment of theinvention, and implanted back into the same mammal from which it wasremoved. The cell can be, but is not required to be, removed from thesame site at which it is implanted. Such embodiments, sometimes known asex vivo gene therapy, can provide a continuous supply of the NGR1polypeptide or polypeptide fragment of the invention, localized at thesite of action, for a limited period of time.

Additional exemplary NgR polypeptides of the invention and methods andmaterials for obtaining these molecules for practicing the presentinvention are described below.

Fusion Proteins and Conjugated Polypeptides

Some embodiments of the invention involve the use of an NgR1 polypeptidethat is not the full-length NgR1 protein, e.g., polypeptide fragments ofNgR1, fused to a heterologous polypeptide moiety to form a fusionprotein. Such fusion proteins can be used to accomplish variousobjectives, e.g., increased serum half-life, improved bioavailability,in vivo targeting to a specific organ or tissue type, improvedrecombinant expression efficiency, improved host cell secretion, ease ofpurification, and higher avidity Depending on the objective(s) to beachieved, the heterologous moiety can be inert or biologically active.Also, it can be chosen to be stably fused to the NgR1 polypeptide moietyof the invention or to be cleavable, in vitro or in vivo. Heterologousmoieties to accomplish these other objectives are known in the art.

In some embodiments of the invention, an NgR1 polypeptide fragment canbe fused to another NgR polypeptide fragment, e.g., an NgR2 or NgR3polypeptide fragment along with Fc.

The human NgR2 polypeptide is shown below as SEQ ID NO:60.

Full-Length Human NgR2 (SEQ ID NO:60):MLPGLRRLLQ APASACLLLM LLALPLAAPS CPMLCTCYSSPPTVSCQANN FSSVPLSLPP STQRLFLQNN LIRTLRPGTFGSNLLTLWLF SNNLSTIYPG TFRHLQALEE LDLGDNRHLRSLEPDTFQGL ERLQSLHLYR CQLSSLPGNI FRGLVSLQYLYLQENSLLHL QDDLFADLAN LSHLFLHGNR LRLLTEHVFRGLGSLDRLLL HGNRLQGVHR AAFRGLSRLT ILYLFNNSLASLPGEALADL PSLEFLRLNA NPWACDCRAR PLWAWFQRARVSSSDVTCAT PPERQGRDLR ALREADFQAC PPAAPTRPGSRARGNSSSNH LYGVAEAGAP PADPSTLYRD LPAEDSRGRQGGDAPTEDDY WGGYGGEDQR GEQMCPGAAG QAPPDSRGPA LSAGLPSPLL CLLLLVPHHL

The mouse NgR2 polypeptide is shown below as SEQ ID NO:61.

Full-Length Mouse NgR2 (SEQ ID NO:61):MLPGLRRLLQ GPASACLLLT LLALPSVTPS CPMLCTCYSSPPTVSCQANN FSSVPLSLPP STQRLFLQNN LIRSLRPGTFGPNLLTLWLF SNNLSTIHPG TFRHLQALEE LDLGDNRHLRSLEPDTFQGL ERLQSLHLYR CQLSSLPGNI FRGLVSLQYLYLQENSLLHL QDDLFADLAN LSHLFLHGNR LRLLTEHVFRGLGSLDRLLL HGNRLQGVHR AAFHGLSRLT ILYLFNNSLASLPGEALADL PALEFLRLNA NPWACDCRAR PLWAWFQRARVSSSDVTCAT PPERQGRDLR ALRDSDFQAC PPPTPTRPGSRARGNSSSNH LYGVAEAGAP PADPSTLYRD LPAEDSRGRQGGDAPTEDDY WGGYGGEDQR GEQTCPGAAC QAPADSRGPA LSAGLRTPLL CLLPLALHHL

The human NgR3 polypeptide is shown below as SEQ ID NO:62.

Full-Length Human NgR3 (SEQ ID NO:62):MLRKGCCVEL LLLLVAAELP LGGGCPRDCV CYPAPMTVSCQAHNFAAIPE GIPVDSERVF LQNNRIGLLQ PGHFSPAMVTLWTYSNNITY IHPSTFEGFV HLEELDLGDN RQLRTLAPETFQGLVKLHAL YLYKCGLSAL PAGVFGGLHS LQYLYLQDNHIEYLQDDIFV DLVNLSHLFL HGNKLWSLGP GTFRGLVNLDRLLLHENQLQ WVHHKAFHDL RRLTTLFLFN NSLSELQGECLAPLGALEFL RLNGNPWDCG CRARSLWEWL QRFRGSSSAVPCVSPGLRHG QDLKLLRAED FRNCTGPASP HQIKSHTLTTTDRAARKEHH SPHGPTRSKG HPHGPRPGHR KPGKNCTNPRNRNQISKAGA GKQAPELPDY APDYQHKFSF DIMPTARPKRKGKCARRTPI RAPSGVQQAS SASSLGASLL AWTLGLAVTL R

The mouse NgR3 polypeptide is shown below as SEQ ID NO:63.

Full-Length Mouse NgR3 (SEQ ID NO:63):MLRKGCCVEL LLLLLAGELP LGGGCPRDGV CYPAPMTVSCQAHNFAAIPE GIPEDSERIF LQNNRITFLQ QGHFSPAMVTLWIYSNNITF LAPNTFEGFV HLEELDLGDN RQLRTLAPETFQGLVKLHAL YLYKCGLSAL PAGIFGGLHS LQYLYLQDNHIEYLQDDIFV DLVNLSHLFL HGNKLWSLGQ GIFRGLVNLDRLLLHENQLQ WVHHKAFHDL HRLTTLFLFN NSLTELQGDCLAPLVALEFL RLNGNAWDCG CRARSLWEWL RRFRGSSSAVPCATPELRQG QDLKLLRVED FRNCTGPVSP HQIKSHTLTTSDRAARKEHH PSHGASRDKG HPHGHPPGSR SGYKKAGKNCTSHRNRNQIS KVSSGKELTE LQDYAPDYQH KFSFDIMPTARPKRKGKCAR RTPIRAPSGV QQASSGTALG APLLAWILGL AVTLR

In some embodiments, the invention provides an isolated polypeptidecomprising a first polypeptide fragment and a second polypeptidefragment, wherein said first polypeptide fragment comprises an aminoacid sequence identical to a first reference amino acid sequence, exceptfor up to one, two, three, four, ten, or twenty individual amino acidsubstitutions, wherein said first reference amino acid sequence isselected from the group consisting of: (a) amino acids a to 305 of SEQID NO:49, (b) amino acids 1 to b of SEQ ID NO:49, and (c) amino acids ato b of SEQ ID NO:49, wherein a is any integer from 1 to 27, and b isany integer from 264 to 309; and wherein said second polypeptidefragment comprises an amino acid sequence identical to a secondreference amino acid sequence, except for up to one, two, three, four,ten, or twenty individual amino acid substitutions, wherein said secondreference amino acid sequence is selected from the group consisting of(a) amino acids c to 332 of SEQ ID NO:60, (b) amino acids 275 to d ofSEQ ID NO:60, and (c) amino acids c to d of SEQ ID NO:60, wherein c isany integer from 265 to 306, and d is any integer from 308 to 340; and;wherein said polypeptide inhibits nogo-receptor-mediated neuriteoutgrowth inhibition.

In one embodiment, the invention provides an isolated polypeptidecomprising a first polypeptide fragment and a second polypeptidefragment, wherein said first polypeptide fragment comprises an aminoacid sequence identical to a first reference amino acid sequence, exceptfor up to one, two, three, four, ten, or twenty individual amino acidsubstitutions, wherein said first reference amino acid sequence is aminoacids 1-274 of SEQ ID NO:49 and wherein said second polypeptide fragmentcomprises an amino acid sequence identical to a second reference aminoacid sequence, except for up to one, two, three, four, ten, or twentyindividual amino acid substitutions, wherein said second reference aminoacid sequence is amino acids 275-311 of SEQ ID NO:60 and; wherein saidpolypeptide inhibits nogo-receptor-mediated neurite outgrowthinhibition.

In one embodiment, the invention provides an isolated polypeptidecomprising a first polypeptide fragment and a second polypeptidefragment, wherein said first polypeptide fragment comprises an aminoacid sequence identical to a first reference amino acid sequence, exceptfor up to one, two, three, four, ten, or twenty individual amino acidsubstitutions, wherein said first reference amino acid sequence is aminoacids 1-274 of SEQ ID NO:49 and wherein said second polypeptide fragmentcomprises an amino acid sequence identical to a second reference aminoacid sequence, except for up to one, two, three, four, ten, or twentyindividual amino acid substitutions, wherein said second reference aminoacid sequence is amino acids 275-332 of SEQ ID NO:60 and; wherein saidpolypeptide inhibits nogo-receptor-mediated neurite outgrowthinhibition.

In one embodiment, the invention provides an isolated polypeptidecomprising a first polypeptide fragment and a second polypeptidefragment, wherein said first polypeptide fragment comprises an aminoacid sequence identical to a first reference amino acid sequence, exceptfor up to one, two, three, four, ten, or twenty individual amino acidsubstitutions, wherein said first reference amino acid sequence is aminoacids 1-305 of SEQ ID NO:49 and wherein said second polypeptide fragmentcomprises an amino acid sequence identical to a second reference aminoacid sequence, except for up to one, two, three, four, ten, or twentyindividual amino acid substitutions, wherein said second reference aminoacid sequence is amino acids 306-311 of SEQ ID NO:60 and; wherein saidpolypeptide inhibits nogo-receptor-mediated neurite outgrowthinhibition.

In one embodiment, the invention provides an isolated polypeptidecomprising a first polypeptide fragment and a second polypeptidefragment, wherein said first polypeptide fragment comprises an aminoacid sequence identical to a first reference amino acid sequence, exceptfor up to one, two, three, four, ten, or twenty individual amino acidsubstitutions, wherein said first reference amino acid sequence is aminoacids 1-305 of SEQ ID NO:49 and wherein said second polypeptide fragmentcomprises an amino acid sequence identical to a second reference aminoacid sequence, except for up to one, two, three, four, ten, or twentyindividual amino acid substitutions, wherein said second reference aminoacid sequence is amino acids 306-309 of SEQ ID NO:60 and; wherein saidpolypeptide inhibits nogo-receptor-mediated neurite outgrowthinhibition. In another embodiment, at least one additional amino acid isadded to the C-terminus of the second polypeptide fragment. Exemplaryamino acids that can be added to the polypeptide include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). In one embodiment, the added amino acid is tryptophan. In anadditional embodiment, the arginine at position 269 of SEQ ID NO:49 issubstituted with tryptophan.

In one embodiment, the invention provides an isolated polypeptidecomprising a first polypeptide fragment and a second polypeptidefragment, wherein said first polypeptide fragment consists of aminoacids 1-310 of SEQ ID NO:49, except for up to one, two, three, four,ten, or twenty individual amino acid substitutions; and wherein saidsecond polypeptide fragment consists of amino acids 311-318 of SEQ IDNO:60 except for up to one, two, three, four, or five individual aminoacid substitutions; and wherein said polypeptide inhibitsnogo-receptor-mediated neurite outgrowth inhibition.

In some embodiments, the polypeptides of the invention further comprisea heterologous polypeptide. In some embodiments, the heterologouspolypeptide is an immunoglobulin constant domain. In some embodiments,the immunoglobulin constant domain is a heavy chain constant domain. Insome embodiments, the heterologous polypeptide is an Fc fragment. Insome embodiments the Fc is joined to the C-terminal end of thepolypeptides of the invention. In some embodiments the fusion is adimer. The invention further encompasses variants, analogs, orderivatives of polypeptide fragments as described above.

As an alternative to expression of a fusion protein, a chosenheterologous moiety can be preformed and chemically conjugated to theNgR polypeptide moiety of the invention. In most cases, a chosenheterologous moiety will function similarly, whether fused or conjugatedto the NgR polypeptide moiety. Therefore, in the following discussion ofheterologous amino acid sequences, unless otherwise noted, it is to beunderstood that the heterologous sequence can be joined to the NgRpolypeptide moiety in the form of a fusion protein or as a chemicalconjugate.

NGR1 aptamers and antibodies and fragments thereof for use in thetreatment methods disclosed herein may also be recombinantly fused to abeterologous polypeptide at the N- or C-terminus or chemicallyconjugated (including covalent and non-covalent conjugations) topolypeptides or other compositions. For example, NgR1 antagonistaptamers and antibodies and fragments thereof may be recombinantly fusedor conjugated to molecules useful as labels in detection assays andeffector molecules such as heterologous polypeptides, drugs,radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387.

NgR1 antagonist polypeptides, aptamers, and antibodies and fragmentsthereof for use in the treatment methods disclosed herein includederivatives that are modified, i.e., by the covalent attachment of anytype of molecule such that covalent attachment does not prevent the NgR1antagonist polypeptide, aptamer, or antibody from inhibiting thebiological function of NgR1. For example, but not by way of limitation,the NgR1 antagonist polypeptides, aptamers and antibodies and fragmentsthereof of the present invention may be modified e.g., by glycosylation,acetylation, pegylation, phosphylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein, etc. Any ofnumerous chemical modifications may be carried out by known techniques,including, but not limited to specific chemical cleavage, acetylation,formylation, metabolic synthesis of tunicamycin, etc. Additionally, thederivative may contain one or more non-classical amino acids.

NgR1 antagonist polypeptides, aptamers and antibodies and fragmentsthereof for use in the treatment methods disclosed herein can becomposed of amino acids joined to each other by peptide bonds ormodified peptide bonds, i.e., peptide isosteres, and may contain aminoacids other than the 20 gene-encoded amino acids. NgR1 antagonistpolypeptides, aptamers and antibodies and fragments thereof may bemodified by natural processes, such as posttranslational processing, orby chemical modification techniques which are well known in the art.Such modifications are well described in basic texts and in moredetailed monographs, as well as in a voluminous research literature.Modifications can occur anywhere in the NgR1 antagonist polypeptide,aptamer or antibody or fragments thereof, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini,or on moieties such as carbohydrates. It will be appreciated that thesame type of modification may be present in the same or varying degreesat several sites in a given NgR1 antagonist polypeptide, aptamer orantibody or fragments thereof. Also, a given NgR1 antagonistpolypeptide, aptamer or antibody or fragments thereof may contain manytypes of modifications. NgR1 antagonist polypeptides, aptamers orantibodies or fragments thereof may be branched, for example, as aresult of ubiquitination, and they may be cyclic, with or withoutbranching. Cyclic, branched, and branched cyclic NgR1 antagonistpolypeptides, aptamers and antibodies or fragments thereof may resultfrom posttranslational natural processes or may be made by syntheticmethods. Modifications include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, pegylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. (See, forinstance, Proteins—Structure And Molecular Properties, T. E. Creighton,W. H. Freeman and Company, New York 2nd Ed., (1993); PosttranslationalCovalent Modification Of Proteins, B. C. Johnson, Ed., Academic Press,New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646(1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992)).

The heterologous polypeptide to which the NgR1 antagonist polypeptide,aptamer or antibody or fragments thereof is fused is usefultherapeutically or is useful to target the NgR1 antagonist polypeptide,aptamer or antibody or fragments thereof. NgR1 antagonist fusionproteins, aptamers and antibodies or fragments thereof can be used toaccomplish various objectives, e.g., increased serum half-life, improvedbioavailability, in vivo targeting to a specific organ or tissue type,improved recombinant expression efficiency, improved host cellsecretion, ease of purification, and higher avidity. Depending on theobjective(s) to be achieved, the heterologous moiety can be inert orbiologically active. Also, it can be chosen to be stably fused to theNgR1 antagonist polypeptide, aptamer or antibody or fragments thereof orto be cleavable, in vitro or in vivo. Heterologous moieties toaccomplish these other objectives are known in the art.

As an alternative to expression of an NgR1 antagonist fusionpolypeptide, aptamer or antibody or fragments thereof, a chosenheterologous moiety can be preformed and chemically conjugated to theantagonist polypeptide, aptamer or antibody. In most cases, a chosenheterologous moiety will function similarly, whether fused or conjugatedto the NgR1 antagonist polypeptide, aptamer or antibody or fragmentsthereof. Therefore, in the following discussion of heterologous aminoacid sequences, unless otherwise noted, it is to be understood that theheterologous sequence can be joined to the NgR1 antagonist polypeptide,aptamer or antibody or fragments thereof in the form of a fusion proteinor as a chemical conjugate.

Pharmacologically active polypeptides such as NgR1 antagonistpolypeptides, aptamers or antibodies or fragments thereof may exhibitrapid in vivo clearance, necessitating large doses to achievetherapeutically effective concentrations in the body. In addition,polypeptides smaller than about 60 kDa potentially undergo glomerularfiltration, which sometimes leads to nephrotoxicity. Fusion orconjugation of relatively small polypeptides such as polypeptidefragments of the NgR signaling domain can be employed to reduce or avoidthe risk of such nephrotoxicity. Various heterologous amino acidsequences, i.e., polypeptide moieties or “carriers,” for increasing thein vivo stability, i.e., serum half-life, of therapeutic polypeptidesare known. Examples include serum albumins such as, e.g., bovine serumalbumin (BSA) or human serum albumin (HSA).

Due to its long half-life, wide in vivo distribution, and lack ofenzymatic or immunological function, essentially full-length human serumalbumin (HSA), or an HSA fragment, is commonly used as a heterologousmoiety. Through application of methods and materials such as thosetaught in Yeh et al., Proc. Natl. Acad. Sci. USA, 89:1904-08 (1992) andSyed et al, Blood 89:3243-52 (1997), HSA can be used to form a fusionprotein or polypeptide conjugate that displays pharmacological activityby virtue of the NgR polypeptide moiety while displaying significantlyincreased in vivo stability, e.g., 10-fold to 100-fold higher. TheC-terminus of the HSA can be fused to the N-terminus of the NgRpolypeptide moiety. Since HSA is a naturally secreted protein, the HSAsignal sequence can be exploited to obtain secretion of the fusionprotein into the cell culture medium when the fusion protein is producedin a eukaryotic, e.g., mammalian, expression system.

In certain embodiments, NgR1 antagonist polypeptides, aptamers,antibodies and antibody fragments thereof for use in the methods of thepresent invention further comprise a targeting moiety. Targetingmoieties include a protein or a peptide which directs localization to acertain part of the body, for example, to the brain or compartmentstherein. In certain embodiments, NgR1 antagonist polypeptides, aptamers,antibodies or antibody fragments thereof for use in the methods of thepresent invention are attached or fused to a brain targeting moiety. Thebrain targeting moieties are attached covalently (e.g., direct,translational fusion, or by chemical linkage either directly or througha spacer molecule, which can be optionally cleavable) or non-covalentlyattached (e.g., through reversible interactions such as avidin:biotin,protein A:IgG, etc.). In other embodiments, the NgR1 antagonistpolypeptides, aptamers, antibodies or antibody fragments thereof for usein the methods of the present invention thereof are attached to one morebrain targeting moieties. In additional embodiments, the brain targetingmoiety is attached to a plurality of NgR1 antagonist polypeptides,aptamers, antibodies or antibody fragments thereof for use in themethods of the present invention.

A brain targeting moiety associated with an NgR1 antagonist polypeptide,aptamer, antibody or antibody fragment thereof enhances brain deliveryof such an NgR1 antagonist polypeptide, antibody or antibody fragmentthereof. A number of polypeptides have been described which, when fusedto a protein or therapeutic agent, delivers the protein or therapeuticagent through the blood brain barrier (BBB). Non-limiting examplesinclude the single domain antibody FC5 (Abulrob et al. (2005) J.Neurochem. 95, 1201-1214); mAB 83-14, a monoclonal antibody to the humaninsulin receptor (Pardridge et al. (1995) Pharmacol. Res. 12, 807-816);the B2, B6 and B8 peptides binding to the human transferrin receptor(hTfR) (Xia et al. (2000) J. Virol. 74, 11359-11366); the OX26monoclonal antibody to the transferrin receptor (Pardridge et al. (1991)J. Pharmacol. Exp. Ther. 259, 66-70); diptheria toxin conjugates (see,for e.g., Gaillard et al., International Congress Series 1277:185-198(2005); and SEQ ID NOs: 1-18 of U.S. Pat. No. 6,306,365. The contents ofthe above references are incorporated herein by reference in theirentirety.

Enhanced brain delivery of an NgR1 composition is determined by a numberof means well established in the art. For example, administering to ananimal a radioactively labelled NgR1 antagonist polypeptide, aptamer,antibody or antibody fragment thereof linked to a brain targetingmoiety; determining brain localization; and comparing localization withan equivalent radioactively labelled NgR1 antagonist polypeptide,aptamer, antibody or antibody fragment thereof that is not associatedwith a brain targeting moiety. Other means of determining enhancedtargeting are described in the above references.

Some embodiments of the invention employ an NgR polypeptide moiety fusedto a hinge and Fc region, i.e., the C-terminal portion of an Ig heavychain constant region. In some embodiments, amino acids in the hingeregion may be substituted with different amino acids. Exemplary aminoacid substitutions for the hinge region according to these embodimentsinclude substitutions of individual cysteine residues in the hingeregion with different amino acids. Any different amino acid may besubstituted for a cysteine in the hinge region. Amino acid substitutionsfor the amino acids of the polypeptides of the invention and thereference amino acid sequence can include amino acids with basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Typical aminoacids to substitute for cysteines in the reference amino acid includealanine, serine, threonine, in particular, serine and alanine. Makingsuch substitutions through engineering of a polynucleotide encoding thepolypeptide fragment is well within the routine expertise of one ofordinary skill in the art.

Potential advantages of an NgR-polypeptide-Fc fusion include solubility,in vivo stability, and multivalency, e.g., dimerization. The Fc regionused can be an IgA, IgD, or IgG Fc region (hinge-CH2-CH3).Alternatively, it can be an IgE or IgM Fc region (hinge-CH2-CH3-CH4). AnIgG Fc region is generally used, e.g., an IgG1 Fc region or IgG4 Fcregion. Materials and methods for constructing and expressing DNAencoding Fc fusions are known in the art and can be applied to obtainfusions without undue experimentation. Some embodiments of the inventionemploy a fusion protein such as those described in Capon et al., U.S.Pat. Nos. 5,428,130 and 5,565,335.

The signal sequence is a polynucleotide that encodes an amino acidsequence that initiates transport of a protein across the membrane ofthe endoplasmic reticulum. Signal sequences useful for constructing animmunofusin include antibody light chain signal sequences, e.g. antibody14.18 (Gillies et al., J. Immunol. Meth., 125:191-202 (1989)), antibodyheavy chain signal sequences, e.g., the MOPC141 antibody heavy chainsignal sequence (Sakano et al., Nature 286:5774 (1980)). Alternatively,other signal sequences can be used. See, e.g. Watson, Nucl. Acids Res.12:5145 (1984). The signal peptide is usually cleaved in the lumen ofthe endoplasmic reticulum by signal peptidases. This results in thesecretion of an immunofusin protein containing the Fc region and the NgRpolypeptide moiety.

In some embodiments, the DNA sequence may encode a proteolytic cleavagesite between the secretion cassette and the NgR polypeptide moiety. Sucha cleavage site may provide, e.g., for the proteolytic cleavage of theencoded fusion protein, thus separating the Fc domain from the targetprotein. Useful proteolytic cleavage sites include amino acid sequencesrecognized by proteolytic enzymes such as trypsin, plasmin, thrombin,factor Xa, or enterokinase K.

The secretion cassette can be incorporated into a replicable expressionvector. Useful vectors include linear nucleic acids, plasmids,phagemids, cosmids and the like. An exemplary expression vector is pdC,in which the transcription of the immunofusin DNA is placed under thecontrol of the enhancer and promoter of the human cytomegalovirus. See,e.g., Lo et al., Biochim. Biophys. Acta 1088:712 (1991); and Lo et al.,Protein Engineering 11:495-500 (1998). An appropriate host cell can betransformed or transfected with a DNA that encodes an NgR1 polypeptideor polypeptide fragment of the invention and used for the expression andsecretion of the polypeptide. Host cells that are typically used includeimmortal hybridoma cells, myeloma cells, 293 cells, Chinese hamsterovary (CHO) cells, Hela cells, and COS cells.

Fully intact, wild-type Fc regions display effector functions thatnormally are unnecessary and undesired in an Fc fusion protein used inthe methods of the present invention. Therefore, certain binding sitestypically are deleted from the Fc region during the construction of thesecretion cassette. For example, since coexpression with the light chainis unnecessary, the binding site for the heavy chain binding protein,Bip (Hendershot et al., Immunol. Today 8:111-14 (1987)), is deleted fromthe CH2 domain of the Fc region of IgE, such that this site does notinterfere with the efficient secretion of the immunofusin. Transmembranedomain sequences, such as those present in IgM, also are generallydeleted.

The IgG1 Fc region is most often used. Alternatively, the Fc region ofthe other subclasses of immunoglobulin gamma (gamma-2, gamma-3 andgamma-4) can be used in the secretion cassette. The IgG1 Fc region ofimmunoglobulin gamma-1 is generally used in the secretion cassette andincludes at least part of the hinge region, the CH2 region, and the CH3region. In some embodiments, the Fc region of immunoglobulin gamma-1 isa CH2-deleted-Fc, which includes part of the hinge region and the CH3region, but not the CH2 region. A CH2-deleted-Fc has been described byGillies et al., Hum. Antibod. Hybridomas 1:47 (1990). In someembodiments, the Fc region of one of IgA, IgD, IgE, or IgM, is used.

NgR-polypeptide-moiety-Fc fusion proteins can be constructed in severaldifferent configurations. In one configuration the C-terminus of the NgRpolypeptide moiety is fused directly to the N-terminus of the Fc hingemoiety. In a slightly different configuration, a short polypeptide, e.g.2-10 amino acids, is incorporated into the fusion between the N-terminusof the NgR polypeptide moiety and the C-terminus of the Fc moiety. Inthe alternative configuration, the short polypeptide is incorporatedinto the fusion between the C-terminus of the NgR polypeptide moiety andthe N-terminus of the Fc moiety. An exemplary embodiment of thisconfiguration is NgR1(310)-2XG4S-Fc, which is amino acids 26-310 of SEQID NO:49 linked to (Gly-Gly-Gly-Gly-Ser)₂ (SEQ ID NO:66) which is linkedto Fc. Such a linker provides conformational flexibility, which mayimprove biological activity in some circumstances. If a sufficientportion of the hinge region is retained in the Fc moiety, theNgR-polypeptide-moiety-Fc fusion will dimerize, thus forming a divalentmolecule. A homogeneous population of monomeric Fc fusions will yieldmonospecific, bivalent dimers. A mixture of two monomeric Fc fusionseach having a different specificity will yield bispecific, bivalentdimers.

Any of a number of cross-linkers that contain a correspondingamino-reactive group and thiol-reactive group can be used to link anNgR1 polypeptide or polypeptide fragment of the invention to serumalbumin. Examples of suitable linkers include amine reactivecross-linkers that insert a thiol-reactive maleimide, e.g., SMCC, AMAS,BMPS, MBS, EMCS, SMPB, SMPH, KMUS, and GMBS. Other suitable linkersinsert a thiol-reactive haloacetate group, e.g., SBAP, SIA, SIAB.Linkers that provide a protected or non-protected thiol for reactionwith sulfhydryl groups to product a reducible linkage include SPDP,SMPT, SATA, and SATP. Such reagents are commercially available (e.g.,Pierce Chemical Company, Rockford, Ill.).

Conjugation does not have to involve the N-terminus of an NgR1polypeptide or polypeptide fragment of the invention or the thiol moietyon serum albumin. For example, NgR-polypeptide-albumin fusions can beobtained using genetic engineering techniques, wherein the NgRpolypeptide moiety is fused to the serum albumin gene at its N-terminus,C-terminus, or both.

NgR polypeptides of the invention can be fused to a polypeptide tag. Theterm “polypeptide tag,” as used herein, is intended to mean any sequenceof amino acids that can be attached to, connected to, or linked to anNgR polypeptide and that can be used to identify, purify, concentrate orisolate the NgR polypeptide. The attachment of the polypeptide tag tothe NgR polypeptide may occur, e.g., by constructing a nucleic acidmolecule that comprises: (a) a nucleic acid sequence that encodes thepolypeptide tag, and (b) a nucleic acid sequence that encodes an NgRpolypeptide. Exemplary polypeptide tags include, e.g., amino acidsequences that are capable of being post-translationally modified, e.g.,amino acid sequences that are biotinylated. Other exemplary polypeptidetags include, e.g., amino acid sequences that are capable of beingrecognized and/or bound by an antibody (or fragment thereof) or otherspecific binding reagent. Polypeptide tags that are capable of beingrecognized by an antibody (or fragment thereof) or other specificbinding reagent include, e.g., those that are known in the art as“epitope tags.” An epitope tag may be a natural or an artificial epitopetag. Natural and artificial epitope tags are known in the art,including, e.g., artificial epitopes such as FLAG, Strep, orpoly-histidine peptides. FLAG peptides include the sequenceAsp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (SEQ ID NO:74) orAsp-Tyr-Lys-Asp-Glu-Asp-Asp-Lys (SEQ ID NO:75) (Einhauer, A. andJungbauer, A., J. Biochem. Biophys. Methods 49:1-3:455-465 (2001)). TheStrep epitope has the sequence Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly (SEQID NO:76). The VSV-G epitope can also be used and has the sequenceTyr-Thr-Asp-11e-Glu-Met-Asn-Arg-Leu-Gly-Lys (SEQ ID NO:77). Anotherartificial epitope is a poly-His sequence having six histidine residues(His-His-His-His-His-His (SEQ ID NO:78). Naturally-occurring epitopesinclude the influenza virus hemagglutinin (HA) sequenceTyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala-Ile-Glu-Gly-Arg (SEQ ID NO:79)recognized by the monoclonal antibody 12CA5 (Murray et al., Anal.Biochem. 229:170-179 (1995)) and the eleven amino acid sequence fromhuman c-myc (Myc) recognized by the monoclonal antibody 9E10(Glu-Gln-Lys-Leu-Leu-Ser-Glu-Glu-Asp-Leu-Asn (SEQ ID NO:80) (Manstein etal., Gene 162:129-134 (1995)). Another useful epitope is the tripeptideGlu-Glu-Phe which is recognized by the monoclonal antibody YL ½.(Stammers et al. FEBS Lett. 283:298-302 (1991)).

In certain embodiments, the NgR polypeptide and the polypeptide tag maybe connected via a linking amino acid sequence. As used herein, a“linking amino acid sequence” may be an amino acid sequence that iscapable of being recognized and/or cleaved by one or more proteases.Amino acid sequences that can be recognized and/or cleaved by one ormore proteases are known in the art. Exemplary amino acid sequences arethose that are recognized by the following proteases: factor VIIa,factor Ixa, factor Xa, APC, t-PA, u-PA, trypsin, chymotrypsin,enterokinase, pepsin, cathepsin B,H,L,S,D, cathepsin G, renin,angiotensin converting enzyme, matrix metalloproteases (collagenases,stromelysins, gelatinases), macrophage elastase, Cir, and Cis. The aminoacid sequences that are recognized by the aforementioned proteases areknown in the art. Exemplary sequences recognized by certain proteasescan be found, e.g., in U.S. Pat. No. 5,811,252.

Polypeptide tags can facilitate purification using commerciallyavailable chromatography media.

In some embodiments of the invention, an NgR polypeptide fusionconstruct is used to enhance the production of an NgR polypeptide moietyin bacteria. In such constructs a bacterial protein normally expressedand/or secreted at a high level is employed as the N-terminal fusionpartner of an NgR1 polypeptide or polypeptide fragment of the invention.See, e.g. Smith et al., Gene 67:31 (1988); Hopp et al., Biotechnology6:1204 (1988); La Vallie et al., Biotechnology 11:187 (1993).

By fusing an NgR polypeptide moiety at the amino and carboxy termini ofa suitable fusion partner, bivalent or tetravalent forms of an NgR1polypeptide or polypeptide fragment of the invention can be obtained.For example, an NgR polypeptide moiety can be fused to the amino andcarboxy termini of an Ig moiety to produce a bivalent monomericpolypeptide containing two NgR polypeptide moieties. Upon dimerizationof two of these monomers, by virtue of the Ig moiety, a tetravalent formof an NgR polypeptide is obtained. Such multivalent forms can be used toachieve increased binding affinity for the target. Multivalent forms ofan NgR1 polypeptide or polypeptide fragment of the invention also can beobtained by placing NgR polypeptide moieties in tandem to formconcatamers, which can be employed alone or fused to a fusion partnersuch as Ig or HSA.

Conjugated Polymers (Other than Polypeptides)

Some embodiments of the invention involve an NgR1 polypeptide orpolypeptide fragment of the invention wherein one or more polymers areconjugated (covalently linked) to the NgR polypeptide. Examples ofpolymers suitable for such conjugation include polypeptides (discussedabove), sugar polymers and polyalkylene glycol chains. Typically, butnot necessarily, a polymer is conjugated to the NgR1 polypeptide orpolypeptide fragment of the invention for the purpose of improving oneor more of the following: solubility, stability, or bioavailability.

The class of polymer generally used for conjugation to an NgR1polypeptide or polypeptide fragment of the invention is a polyalkyleneglycol. Polyethylene glycol (PEG) is most frequently used. PEG moieties,e.g., 1, 2, 3, 4 or 5 PEG polymers, can be conjugated to each NgRpolypeptide to increase serum half life, as compared to the NgRpolypeptide alone. PEG moieties are non-antigenic and essentiallybiologically inert. PEG moieties used in the practice of the inventionmay be branched or unbranched.

The number of PEG moieties attached to the NgR polypeptide and themolecular weight of the individual PEG chains can vary. In general, thehigher the molecular weight of the polymer, the fewer polymer chainsattached to the polypeptide. Usually, the total polymer mass attached toan NgR polypeptide or polypeptide fragment is from 20 kDa to 40 kDa.Thus, if one polymer chain is attached, the molecular weight of thechain is generally 20-40 kDa. If two chains are attached, the molecularweight of each chain is generally 10-20 kDa. If three chains areattached, the molecular weight is generally 7-14 kDa.

The polymer, e.g., PEG, can be linked to the NgR polypeptide through anysuitable, exposed reactive group on the polypeptide. The exposedreactive group(s) can be, e.g., an N-terminal amino group or the epsilonamino group of an internal lysine residue, or both. An activated polymercan react and covalently link at any free amino group on the NgRpolypeptide. Free carboxylic groups, suitably activated carbonyl groups,hydroxyl, guanidyl, imidazole, oxidized carbohydrate moieties andmercapto groups of the NgR polypeptide (if available) also can be usedas reactive groups for polymer attachment.

In a conjugation reaction, from about 1.0 to about 10 moles of activatedpolymer per mole of polypeptide, depending on polypeptide concentration,is typically employed. Usually, the ratio chosen represents a balancebetween maximizing the reaction while minimizing side reactions (oftennon-specific) that can impair the desired pharmacological activity ofthe NgR polypeptide moiety. Preferably, at least 50% of the biologicalactivity (as demonstrated, e.g., in any of the assays described hereinor known in the art) of the NgR polypeptide is retained, and mostpreferably nearly 100% is retained.

The polymer can be conjugated to the NgR polypeptide using conventionalchemistry. For example, a polyalkylene glycol moiety can be coupled to alysine epsilon amino group of the NgR polypeptide. Linkage to the lysineside chain can be performed with an N-hydroxysuccinimide (NHS) activeester such as PEG succinimidyl succinate (SS-PEG) and succinimidylpropionate (SPA-PEG). Suitable polyalkylene glycol moieties include,e.g., carboxymethyl-NHS and norleucine-NHS, SC. These reagents arecommercially available. Additional amine-reactive PEG linkers can besubstituted for the succinimidyl moiety. These include, e.g.,isothiocyanates, nitrophenylcarbonate (PNP), epoxides, benzotriazolecarbonates, SC-PEG, tresylate, aldehyde, epoxide, carbonylimidazole andPNP carbonate. Conditions are usually optimized to maximize theselectivity and extent of reaction. Such optimization of reactionconditions is within ordinary skill in the art.

PEGylation can be carried out by any of the PEGylation reactions knownin the art. See, e.g., Focus on Growth Factors, 3: 4-10, 1992 andEuropean patent applications EP 0 154 316 and EP 0 401 384. PEGylationmay be carried out using an acylation reaction or an alkylation reactionwith a reactive polyethylene glycol molecule (or an analogous reactivewater-soluble polymer).

PEGylation by acylation generally involves reacting an active esterderivative of polyethylene glycol. Any reactive PEG molecule can beemployed in the PEGylation. PEG esterified to N-hydroxysuccinimide (NHS)is a frequently used activated PEG ester. As used herein, “acylation”includes without limitation the following types of linkages between thetherapeutic protein and a water-soluble polymer such as PEG: amide,carbamate, urethane, and the like. See, e.g., Bioconjugate Chem. 5:133-140, 1994. Reaction parameters are generally selected to avoidtemperature, solvent, and pH conditions that would damage or inactivatethe NgR polypeptide.

Generally, the connecting linkage is an amide and typically at least 95%of the resulting product is mono-, di- or tri-PEGylated. However, somespecies with higher degrees of PEGylation may be formed in amountsdepending on the specific reaction conditions used. Optionally, purifiedPEGylated species are separated from the mixture, particularly unreactedspecies, by conventional purification methods, including, e.g.,dialysis, salting-out, ultrafiltration, ion-exchange chromatography, gelfiltration chromatography, hydrophobic exchange chromatography, andelectrophoresis.

PEGylation by alkylation generally involves reacting a terminal aldehydederivative of PEG with an NgR1 polypeptide or polypeptide fragment ofthe invention in the presence of a reducing agent. In addition, one canmanipulate the reaction conditions to favor PEGylation substantiallyonly at the N-terminal amino group of the NgR polypeptide, i.e. amono-PEGylated protein. In either case of mono-PEGylation orpoly-PEGylation, the PEG groups are typically attached to the proteinvia a —CH2-NH— group. With particular reference to the —CH2- group, thistype of linkage is known as an “alkyl” linkage.

Derivatization via reductive alkylation to produce an N-terminallytargeted mono-PEGylated product exploits differential reactivity ofdifferent types of primary amino groups (lysine versus the N-terminal)available for derivatization. The reaction is performed at a pH thatallows one to take advantage of the pKa differences between theepsilon-amino groups of the lysine residues and that of the N-terminalamino group of the protein. By such selective derivatization, attachmentof a water-soluble polymer that contains a reactive group, such as analdehyde, to a protein is controlled: the conjugation with the polymertakes place predominantly at the N-terminus of the protein and nosignificant modification of other reactive groups, such as the lysineside chain amino groups, occurs.

The polymer molecules used in both the acylation and alkylationapproaches are selected from among water-soluble polymers. The polymerselected is typically modified to have a single reactive group, such asan active ester for acylation or an aldehyde for alkylation, so that thedegree of polymerization may be controlled as provided for in thepresent methods. An exemplary reactive PEG aldehyde is polyethyleneglycol propionaldehyde, which is water stable, or mono C1-C10 alkoxy oraryloxy derivatives thereof (see, e.g., Harris et al., U.S. Pat. No.5,252,714). The polymer may be branched or unbranched. For the acylationreactions, the polymer(s) selected typically have a single reactiveester group. For reductive alkylation, the polymer(s) selected typicallyhave a single reactive aldehyde group. Generally, the water-solublepolymer will not be selected from naturally occurring glycosyl residues,because these are usually made more conveniently by mammalianrecombinant expression systems.

Methods for preparing a PEGylated NgR polypeptides of the inventiongenerally includes the steps of (a) reacting an NgR1 polypeptide orpolypeptide fragment of the invention with polyethylene glycol (such asa reactive ester or aldehyde derivative of PEG) under conditions wherebythe molecule becomes attached to one or more PEG groups, and (b)obtaining the reaction product(s). In general, the optimal reactionconditions for the acylation reactions will be determined case-by-casebased on known parameters and the desired result. For example, a largerthe ratio of PEG to protein, generally leads to a greater the percentageof poly-PEGylated product.

Reductive alkylation to produce a substantially homogeneous populationof mono-polymer/NgR polypeptide generally includes the steps of: (a)reacting an NgR1 polypeptide or polypeptide fragment of the inventionwith a reactive PEG molecule under reductive alkylation conditions, at apH suitable to permit selective modification of the N-terminal aminogroup of NgR; and (b) obtaining the reaction product(s).

For a substantially homogeneous population of mono-polymer/NgRpolypeptide, the reductive alkylation reaction conditions are those thatpermit the selective attachment of the water-soluble polymer moiety tothe N-terminus of an NgR1 polypeptide or polypeptide fragment of theinvention. Such reaction conditions generally provide for pKadifferences between the lysine side chain amino groups and theN-terminal amino group. For purposes of the present invention, the pH isgenerally in the range of 3-9, typically 3-6.

NgR polypeptides of the invention can include a tag, e.g., a moiety thatcan be subsequently released by proteolysis. Thus, the lysine moiety canbe selectively modified by first reacting a His-tag modified with alow-molecular-weight linker such as Traut's reagent (Pierce ChemicalCompany, Rockford, Ill.) which will react with both the lysine andN-terminus, and then releasing the His tag. The polypeptide will thencontain a free SH group that can be selectively modified with a PEGcontaining a thiol-reactive head group such as a maleimide group, avinylsulfone group, a haloacetate group, or a free or protected SH.

Traut's reagent can be replaced with any linker that will set up aspecific site for PEG attachment. For example, Traut's reagent can bereplaced with SPDP, SMPT, SATA, or SATP (Pierce Chemical Company,Rockford, Ill.). Similarly one could react the protein with anamine-reactive linker that inserts a maleimide (for example SMCC, AMAS,BMPS, MBS, EMCS, SMPB, SMPH, KMUS, or GMBS), a haloacetate group (SBAP,SIA, SIAB), or a vinylsulfone group and react the resulting product witha PEG that contains a free SH.

In some embodiments, the polyalkylene glycol moiety is coupled to acysteine group of the NgR polypeptide. Coupling can be effected using,e.g. a maleimide group, a vinylsulfone group, a haloacetate group, or athiol group.

Optionally, the NgR polypeptide is conjugated to the polyethylene-glycolmoiety through a labile bond. The labile bond can be cleaved in, e.g.,biochemical hydrolysis, proteolysis, or sulfhydryl cleavage. Forexample, the bond can be cleaved under in vivo (physiological)conditions.

The reactions may take place by any suitable method used for reactingbiologically active materials with inert polymers, generally at about pH5-8, e.g. pH 5, 6, 7, or 8, if the reactive groups are on the alphaamino group at the N-terminus. Generally the process involves preparingan activated polymer and thereafter reacting the protein with theactivated polymer to produce the soluble protein suitable forformulation.

The NgR polypeptides of the invention, in certain embodiments, aresoluble polypeptides. Methods for making a polypeptide soluble orimproving the solubility of a polypeptide are well known in the art.

Nucleic Acid Molecules of the Present Invention

The present invention provide a nucleic acid that encodes a polypeptideof the invention, including the polypeptides of any one of SEQ ID NOs:1-9, 26-27, 29-37 and 41-45. In some embodiments, the nucleic acidencodes a polypeptide selected from the group consisting of amino acidresidues 26-344 of Nogo receptor-1 as shown in SEQ ID NOs: 6 and 8 oramino acid residues 27-344 of Nogo receptor-1 as shown in SEQ ID NO: 8.In some embodiments, the nucleic acid molecule encodes a polypeptideselected from the group consisting of amino acid residues 26-310 of Nogoreceptor-1 as shown in SEQ ID NOs: 7 and 9 or amino acid residues 27-310of Nogo receptor-1 as shown in SEQ ID NO: 9. As used herein, “nucleicacid” means genomic DNA, cDNA, mRNA and antisense molecules, as well asnucleic acids based on alternative backbones or including alternativebases whether derived from natural sources or synthesized. In someembodiments, the nucleic acid further comprises a transcriptionalpromoter and optionally a signal sequence each of which is operablylinked to the nucleotide sequence encoding the polypeptides of theinvention.

In some embodiments, the invention provides a nucleic acid encoding aNogo receptor-1 fusion protein of the invention, including a fusionprotein comprising a polypeptide selected from the group consisting ofamino acid residues 26-344 of Nogo receptor-1 as shown in SEQ ID NOs: 6and 8 or amino acid residues 27-344 of SEQ ID NO: 8 and amino acidresidues 26-310 of Nogo receptor-1 as shown in SEQ ID NOs: 7 and 9 oramino acid residues 27-310 of SEQ ID NO: 9. In some embodiments, thenucleic acid encodes a Nogo receptor-1 fusion protein comprising apolypeptides selected from the group consisting of SEQ ID NOs: 26-27,29-37 and 41-45. In some embodiments, the nucleic acid encoding a Nogoreceptor-1 fusion protein further comprises a transcriptional promoterand optionally a signal sequence. In some embodiments, the nucleotidesequence further encodes an immunoglobulin constant region. In someembodiments, the immunoglobulin constant region is a heavy chainconstant region. In some embodiments, the nucleotide sequence furtherencodes an immunoglobulin heavy chain constant region joined to a hingeregion. In some embodiments the nucleic acid further encodes Fc. In someembodiments the Nogo receptor-1 fusion proteins comprise an Fc fragment.

The encoding nucleic acids of the present invention may further bemodified so as to contain a detectable label for diagnostic and probepurposes. A variety of such labels are known in the art and can readilybe employed with the encoding molecules herein described. Suitablelabels include, but are not limited to, biotin, radiolabeled nucleotidesand the like. A skilled artisan can employ any of the art known labelsto obtain a labeled encoding nucleic acid molecule.

The present invention also includes polynucleotides that hybridize undermoderately stringent or high stringency conditions to the noncodingstrand, or complement, of a polynucleotide that encodes any one of thepolypeptides of the invention. Stringent conditions are known to thoseskilled in the art and can be found in CURRENT PROTOCOLS IN MOLECULARBIOLOGY, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.

The human Nogo receptor-1 polynucleotide is shown below as SEQ ID NO:81.

Full-Length Human Nogo receptor-1 is encoded by nucleotide 166 tonucleotide 1587 of SEQ ID NO:81:

agcccagcca gagccgggcg gagcggagcg cgccgagcct cgtcccgcgg ccgggccggggccgggccgt agcggcggcg cctggatgcg gacccggccg cggggagacg ggcgcccgccccgaaacgac tttcagtccc cgacgcgccc cgcccaaccc ctacgatgaa gagggcgtccgctggaggga gccggctgct ggcatgggtg ctgtggctgc aggcctggca ggtggcagccccatgcccag gtgcctgcgt atgctacaat gagcccaagg tgacgacaag ctgcccccagcagggcctgc aggctgtgcc cgtgggcatc cctgctgcca gccagcgcat cttcctgcacggcaaccgca tctcgcatgt gccagctgcc agcttccgtg cctgccgcaa cctcaccatcctgtggctgc actcgaatgt gctggcccga attgatgcgg ctgccttcac tggcctggccctcctggagc agctggacct cagcgataat gcacagctcc ggtctgtgga ccctgccacattccacggcc tgggccgcct acacacgctg cacctggacc gctgcggcct gcaggagctgggcccggggc tgttccgcgg cctggctgcc ctgcagtacc tctacctgca ggacaacgcgctgcaggcac tgcctgatga caccttccgc gacctgggca acctcacaca cctcttcctgcacggcaacc gcatctccag cgtgcccgag cgcgccttcc gtgggctgca cagcctcgaccgtctcctac tgcaccagaa ccgcgtggcc catgtgcacc cgcatgcctt ccgtgaccttggccgcctca tgacactcta tctgtttgcc aacaatctat cagcgctgcc cactgaggccctggcccccc tgcgtgccct gcagtacctg aggctcaacg acaacccctg ggtgtgtgactgccgggcac gcccactctg ggcctggctg cagaagttcc gcggctcctc ctccgaggtgccctgcagcc tcccgcaacg cctggctggc cgtgacctca aacgcctagc tgccaatgacctgcagggct gcgctgtggc caccggccct taccatccca tctggaccgg cagggccaccgatgaggagc cgctggggct tcccaagtgc tgccagccag atgccgctga caaggcctcagtactggagc ctggaagacc agcttcggca ggcaatgcgc tgaagggacg cgtgccgcccggtgacagcc cgccgggcaa cggctctggc ccacggcaca tcaatgactc accctttgggactctgcctg gctctgctga gcccccgctc actgcagtgc ggcccgaggg ctccgagccaccagggttcc ccacctcggg ccctcgccgg aggccaggct gttcacgcaa gaaccgcacccgcagccact gccgtctggg ccaggcaggc agcgggggtg gcgggactgg tgactcagaaggctcaggtg ccctacccag cctcacctgc agcctcaccc ccctgggcct ggcgctggtgctgtggacag tgcttgggcc ctgctgaccc ccagcggaca caagagcgtg ctcagcagccaggtgtgtgt acatacgggg tctctctcca cgccgccaag ccagccgggc ggccgacccgtggggcaggc caggccaggt cctccctgat ggacgcctg

The rat Nogo receptor-1 polynucleotide is shown below as SEQ ID NO:82and is accession number NM_(—)053613 in Genbank.

atgaagaggg cgtcctccgg aggaagccgg ctgccgacat gggtgttatg gctacaggcctggagggtag caacgccctg ccctggtgcc tgtgtgtgct acaatgagcc caaggtcacaacaagccgcc cccagcaggg cctgcaggct gtacccgctg gcatcccagc ctccagccagagaatcttcc tgcacggcaa ccgaatctct tacgtgccag ccgccagctt ccagtcatgccggaatctca ccatcctgtg gctgcactca aatgcgctgg ccgggattga tgccgcggccttcactggtc tgaccctcct ggagcaacta gatcttagtg acaatgcaca gctccgtgtcgtggacccca ccacgttccg tggcctgggc cacctgcaca cgctgcacct agaccgatgcggcctgcagg agctggggcc tggcctattc cgtgggctgg cagctctgca gtacctctacctacaagaca acaacctgca ggcacttccc gacaacacct tccgagacct gggcaacctcacgcatctct ttctgcatgg caaccgtatc cccagtgttc ctgagcacgc tttccgtggcttgcacagtc ttgaccgtct cctcttgcac cagaaccatg tggctcgtgt gcacccacatgccttccggg accttggccg actcatgacc ctctacctgt ttgccaacaa cctctccatgctccccgcag aggtcctagt gcccctgagg tctctgcagt acctgcgact caatgacaacccctgggtgt gtgactgcag ggcacgtccg ctctgggcct ggctgcagaa gttccgaggttcctcatccg gggtgcccag caacctaccc caacgcctgg caggccgtga tctgaagcgcctggctacca gtgacttaga gggttgtgct gtggcttcgg ggcccttccg tcccttccagaccaatcagc tcactgatga ggagctgctg ggcctcccca agtgctgcca gccggatgctgcagacaagg cctcagtact ggaacccggg aggccggcgt ctgttggaaa tgcactcaagggacgtgtgc ctcccggtga cactccacca ggcaatggct caggcccacg gcacatcaatgactctccat ttgggacttt gcccggctct gcagagcccc cactgactgc cctgcggcctgggggttccg agcccccggg actgcccacc acgggccccc gcaggaggcc aggttgttccagaaagaacc gcacccgtag ccactgccgt ctgggccagg caggaagtgg gagcagtggaactggggatg cagaaggttc gggggccctg cctgccctgg cctgcagcct tgctcctctgggccttgcac tggtactttg gacagtgctt gggccctgct ga

The mouse Nogo receptor-1 polynucleotide is shown below as SEQ ID NO:83and is accession number NM_(—)022982 in Genbank.

agccgcagcc cgcgagccca gcccggcccg gtagagcgga gcgccggagc ctcgtcccgcggccgggccg ggaccgggcc ggagcagcgg cgcctggatg cggacccggc cgcgcgcagacgggcgcccg ccccgaagcc gcttccagtg cccgacgcgc cccgctcgac cccgaagatgaagagggcgt cctccggagg aagcaggctg ctggcatggg tgttatggct acaggcctggagggtagcaa caccatgccc tggtgcttgt gtgtgctaca atgagcccaa ggtaacaacaagctgccccc agcagggtct gcaggctgtg cccactggca tcccagcctc tagccagcgaatcttcctgc atggcaaccg aatctctcac gtgccagctg cgagcttcca gtcatgccgaaatctcacta tcctgtggct gcactctaat gcgctggctc ggatcgatgc tgctgccttcactggtctga ccctcctgga gcaactagat cttagtgata atgcacagct tcatgtcgtggaccctacca cgttccacgg cctgggccac ctgcacacac tgcacctaga ccgatgtggcctgcgggagc tgggtcccgg cctattccgt ggactagcag ctctgcagta cctctacctacaagacaaca atctgcaggc actccctgac aacacctttc gagacctggg caacctcacgcatctctttc tgcatggcaa ccgtatcccc agtgtgcctg agcacgcttt ccgtggcctgcacagtcttg accgcctcct cttgaaccag aaccatgtgg ctcgtgtgca cccacatgccttccgggacc ttggccgcct catgaccctc tacctgtttg ccaacaacct ctccatgctgcctgcagagg tcctaatgcc cctgaggtct ctgcagtacc tgcgactcaa tgacaacccctgggtgtgtg actgccgggc acgtccactc tgggcctggc tgcagaagtt ccgaggttcctcatcagagg tgccctgcaa cctgccccaa cgcctggcag accgtgatct taagcgcctcgctgccagtg acctagaggg ctgtgctgtg gcttcaggac ccttccgtcc catccagaccagtcagctca ctgatgagga gctgctgagc ctccccaagt gctgccagcc agatgctgcagacaaagcct cagtactgga acccgggagg ccagcttctg ccggaaacgc cctcaagggacgtgtgcctc ccggtgacac tccaccaggc aatggctcag gccctcggca catcaatgactctccatttg gaactttgcc cagctctgca gagcccccac tgactgccct gcggcctgggggttccgagc caccaggact tcccaccact ggtccccgca ggaggccagg ttgttcccggaagaatcgca cccgcagcca ctgccgtctg ggccaggcgg gaagtggggc cagtggaacaggggacgcag agggttcagg ggctctgcct gctctggcct gcagccttgc tcctctgggccttgcactgg tactttggac agtgcttggg ccctgctgac cagccaccag ccaccaggtgtgtgtacata tggggtctcc ctccacgccg ccagccagag ccagggacag gctctgaggggcaggccagg ccctccctga cagatgcctc cccaccagcc cacccccatc tccaccccatcatgtttaca gggttccggg ggtggcgttt gttccagaac gccacctccc acccggatcgcggtatatag agatatgaat tttattttac ttgtgtaaaa tatcggatga cgtggaataaagagctcttt tcttaaaaaa aaaaaaaaaa aa

The human Nogo receptor-2 polynucleotide is shown below as SEQ ID NO:84and is accession number BK001302 in Genbank.

atgctgcccg ggctcaggcg cctgctgcaa gctcccgcct cggcctgcct cctgctgatgctcctggccc tgcccctggc ggcccccagc tgccccatgc tctgcacctg ctactcatccccgcccaccg tgagctgcca ggccaacaac ttctcctctg tgccgctgtc cctgccacccagcactcagc gactcttcct gcagaacaac ctcatccgca cgctgcggcc aggcacctttgggtccaacc tgctcaccct gtggctcttc tccaacaacc tctccaccat ctacccgggcactttccgcc acttgcaagc cctggaggag ctggacctcg gtgacaaccg gcacctgcgctcgctggagc ccgacacctt ccagggcctg gagcggctgc agtcgctgca tttgtaccgctgccagctca gcagcctgcc cggcaacatc ttccgaggcc tggtcagcct gcagtacctctacctccagg agaacagcct gctccaccta caggatgact tgttcgcgga cctggccaacctgagccacc tcttcctcca cgggaaccgc ctgcggctgc tcacagagca cgtgtttcgcggcctgggca gcctggaccg gctgctgctg cacgggaacc ggctgcaggg cgtgcaccgcgcggccttcc gcggcctcag ccgcctcacc atcctctacc tgttcaacaa cagcctggcctcgctgcccg gcgaggcgct cgccgacctg ccctcgctcg agttcctgcg gctcaacgctaacccctggg cgtgcgactg ccgcgcgcgg ccgctctggg cctg9ttcca gcgcgcgcgcgtgtccagct ccgacgtgac ctgcgccacc cccccggagc gccagggccg agacctgcgcgcgctccgcg aggccgactt ccaggcgtgt ccgcccgcgg cacccacgcg gccgggcagccgcgcccgcg gcaacagctc ctccaaccac ctgtacgggg tggccgaggc cggggcgcccccagccgatc cctccaccct ctaccgagat ctgcctgccg aagactcgcg ggggcgccagggcggggacg cgcctactga ggacgactac tgggggggct acgggggtga ggaccagcgaggggagcaga tgtgccccgg cgctgcctgc caggcgcccc cggactcccg aggccctgcgctctcggccg ggctccccag ccctctgctt tgcctcctgc tcctggtgcc ccaccacctc tga

The mouse Nogo receptor-2 polynucleotide is shown below as SEQ ID NO:85and is accession number NM_(—)199223 in Genbank.

atgctgcccg ggctccggcg cctgctgcaa ggtcctgcct cagcctgcct actgctgacactcctggccc ttccttccgt gacccccagc tgtcctatgc tctgcacctg ctactcctccccgcccaccg tgagctgcca ggccaacaac ttctcctcag tgccgctgtc cttgccacccagtacacaga gactcttctt gcagaacaac ctcatccgct cactgcggcc aggcacctttgggcccaacc tgctcaccct gtggctcttc tccaacaacc tctccaccat ccaccctggcaccttccgcc acctgcaggc cctagaagaa ctggacctcg gtgacaaccg gcacctgcgctccctggagc ccgacacctt ccagggtctg gagaggctgc agtcactaca cctgtatcgttgccagctca gcagcctgcc tggcaacatt ttccgaggct tggtcagcct acagtacctctacctccagg agaacagcct gctccatcta caggatgact tgttcgcgga cctggccaacctgagccacc tcttcctcca cgggaaccgc ctgcggctgc tcacggagca cgtgttccgcggcttgggca gcctggaccg gctgttgctg cacgggaacc ggctgcaggg cgtgcaccgcgcggctttcc acggcctcag ccgcctcacc atcctctacc tgttcaacaa cagcctggcctcgctgccgg gagaggcgct ggccgacctg ccggcgctcg agttcctgcg gctcaacgccaacccctggg cgtgcgactg ccgcgctcgg ccgctctggg cttggttcca gcgcgcgcgggtgtccagct ccgacgtgac ctgcgccacc ccgcccgagc gccagggccg ggacctgcgcgcgctgcgcg actccgattt ccaagcgtgc ccgccgccca cgcccacgcg gccgggcagccgcgcccgcg gcaacagctc ttccaaccac ctgtacggcg tggccgaggc tggcgctccccccgcagacc cgtccacgct ctaccgagat ctgcccgccg aggactcgcg ggggcgccagggcggggacg cgcccaccga ggacgactac tgggggggct acggcggcga ggatcagcggggcgagcaga cgtgtcccgg ggccgcgtgc caggcgcccg cagactcgcg tggccccgcgctctcggccg ggctgcgcac ccctctgctc tgcctcttgc ccctggcgct ccatcacctctga

The human Nogo receptor-3 polynucleotide is shown below as SEQ ID NO:86and is accession number BK001305 in Genbank.

atgcttcgca aagggtgctg tgtggagttg ctgctgctgt tggtagctgc ggagctgcccctgggtggtg gctgcccacg ggactgtgtg tgctacccgg cgcccatgac ggtcagctgccaggcgcaca actttgcagc catcccggag ggcatccccg tggacagcga gcgcgtcttcctgcagaaca accgcatcgg cctcctccag cccggccact tcagccccgc catggtcaccctgtggatct actcgaacaa catcacctac atccacccca gcaccttcga gggcttcgtgcacctggagg agctggacct cggcgacaac cggcagctgc ggacgctggc acccgagaccttccagggcc tggtgaagct tcacgccctc tacctctaca agtgtgggct cagcgccttgccggccggcg tctttggcgg cctgcacagc ctgcagtacc tctacctgca ggacaaccacatcgagtacc tccaggacga catcttcgtg gacctggtca acctcagcca cctgtttctccacggcaaca agctgtggag tctgggcccg ggcaccttcc ggggcctggt gaacctggaccgtcttttgc tgcacgagaa ccagctgcag tgggtccacc acaaggcatt ccacgacctccgcaggctga ccaccctctt cctcttcaac aacagcctct cggagctgca gggtgagtgcctggccccgc tgggggccct ggagttcctc cgcctcaatg gcaacccctg ggactgtggttgtcgcgcgc gctccctgtg ggaatggctg cagaggttcc ggggctccag ctccgctgtcccctgtgtgt cccctgggct gcggcacggc caggacctga agctgctgag ggccgaggacttccggaact gcacgggacc agcgtccccg caccagatca agtcacacac gctcaccaccaccgacaggg ccgcccgcaa ggaacaccac tcaccccacg gccccaccag gagcaagggccacccgcacg gcccccggcc cggccacagg aagccgggga agaactgcac caaccccaggaaccgcaatc agatctctaa ggcgggcgcc gggaaacagg cccccgagct gccagactatgccccagact accagcacaa gttcagtttt gacatcatgc ctacggcccg gcccaagaggaagggcaagt gtgcccgcag gacccccatc cgtgccccca gcggggtgca gcaggcctcctcggccagtt ccctgggggc ctccctcctg gcctggacac tggggctggc ggtcactctc cgctga

The mouse Nogo receptor-3 polynucleotide is shown below as SEQ ID NO:87and is accession number BK001304 in Genbank.

atgcttcgca aagggtgctg tgtggaattg ctgctgttgc tgctcgctgg agagctacctctgggtggtg gttgtcctcg agactgtgtg tgctaccctg cgcccatgac tgtcagctgccaggcacaca actttgctgc catcccggag ggcatcccag aggacagtga gcgcatcttcctgcagaaca atcgcatcac cttcctccag cagggccact tcagccccgc catggtcaccctctggatct actccaacaa catcactttc attgctccca acaccttcga gggctttgtgcatctggagg agctagacct tggagacaac cgacagctgc gaacgctggc acccgagaccttccaaggcc tggtgaagct tcacgccctc tacctctata agtgtggact gagcgccctgcccgcaggca tctttggtgg cctgcacagc ctgcagtatc tctacttgca ggacaaccatatcgagtacc tccaagatga catctttgtg gacctggtca atctcagtca cttgtttctccatggtaaca agctatggag cctgggccaa ggcatcttcc ggggcctggt gaacctggaccggttgctgc tgcatgagaa ccagctacag tgggttcacc acaaggcttt ccatgacctccacaggctaa ccaccctctt tctcttcaac aacagcctca ctgagctgca gggtgactgtctggcccccc tggtggcctt ggagttcctt cgcctcaatg ggaatgcttg ggactgtggctgccgggcac gttccctgtg ggaatggctg cgaaggttcc gtggctctag ctctgctgtcccctgcgcga cccccgagct gcggcaaggc caggatctga agctgctgag ggtggaggacttccggaact gcacaggacc agtgtctcct caccagatca agtctcacac gcttaccacctctgacaggg ctgcccgcaa ggagcaccat ccgtcccatg gggcctccag ggacaaaggccacccacatg gccatccgcc tggctccagg tcaggttaca agaaggcagg caagaactgcaccagccaca ggaaccggaa ccagatctct aaggtgagct ctgggaaaga gcttaccgaactgcaggact atgcccccga ctatcagcac aagttcagct ttgacatcat gcccaccgcacgacccaaga ggaagggcaa gtgtgctcgc aggaccccca tccgtgcccc cagtggggtgcagcaggcat cctcaggcac ggcccttggg gccccactcc tggcctggat actggggctggcagtcactc tccgctga

NgR1 Polynucleotide Antagonists

Specific embodiments comprise NgR1 polynucleotide antagonists whichprevent expression of NgR1 (knockdown). NgR1 polynucleotide antagonistsinclude, but are not limited to antisense molecules, ribozymes, siRNA,shRNA and RNAi. Typically, such binding molecules are separatelyadministered to the animal (see, for example, O'Connor, J. Neurochem.56:560 (1991), but such binding molecules may also be expressed in vivofrom polynucleotides taken up by a host cell and expressed in vivo. Seealso Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression,CRC Press, Boca Raton, Fla. (1988).

Expression of the NgR gene can, in some embodiments, be inhibited usingRNA interference (“RNAi”). RNAi refers to the expression of an RNA whichinterferes with the expression of the targeted mRNA. RNAi is aphenomenon in which the introduction of double-stranded RNA (dsRNA) intoa cell causes degradation of the homologous mRNA. First discovered inthe nematode Caenorhabditis elegans, RNAi has since been found tooperate in a wide range of organisms. An “RNAi nucleic acid” as usedherein is a nucleic acid sequence generally shorter than 50 nucleotidesin length, that causes gene silencing at the mRNA level.

For example, in mammalian cells, introduction of long dsRNA (>30nucleotides) can initiate a potent antiviral response, exemplified bynonspecific inhibition of protein synthesis and RNA degradation. RNAinterference provides a mechanism of gene silencing at the mRNA level.In recent years, RNAi has become an endogenous and potent gene-specificsilencing technique that uses double-stranded RNAs (dsRNA) to mark aparticular transcript for degradation in vivo. It also offers anefficient and broadly applicable approach for gene knock-out. Inaddition, RNAi technology can be used for therapeutic purposes. Forexample, RNAi targeting Fas-mediated apoptosis has been shown to protectmice from fulminant hepatitis. RNAi technology has been disclosed innumerous publications, such as U.S. Pat. Nos. 5,919,619, 6,506,559 andPCT Publication Nos. WO99/14346, WO01/70949, WO01/36646, WO00/63364,WO00/44895, WO01/75164, WO01/92513, WO01/68836 and WO01/29058.

Specifically, the RNAi silences a targeted gene via interacting with thespecific mRNA (e.g. NgR1) through a siRNA (short interfering RNA). Theds RNA complex is then targeted for degradation by the cell. AdditionalRNAi molecules include Short hairpin RNA (shRNA); also short interferinghairpin. The shRNA molecule contains sense and antisense sequences froma target gene connected by a loop. The shRNA is transported from thenucleus into the cytoplasm, it is degraded along with the mRNA. Pol IIIor U6 promoters can be used to express RNAs for RNAi. A sequence capableof inhibiting gene expression by RNA interference can have any length.For instance, the sequence can have at least 10, 15, 20, 25, 30, 35, 40,45, 50, 100 or more consecutive nucleotides. The sequence can be dsRNAor any other type of polynucleotide, provided that the sequence can forma functional silencing complex to degrade the target mRNA transcript.

RNAi is mediated by double stranded RNA (dsRNA) molecules that havesequence-specific homology to their “target” mRNAs (Caplen et al., ProcNatl Acad Sci USA 98:9742-9747, 2001). Biochemical studies in Drosophilacell-free lysates indicates that the mediators of RNA-dependent genesilencing are 18-25 nucleotide “small interfering” RNA duplexes(siRNAs). Accordingly, siRNA molecules are advantageously used in themethods of the present invention. siRNAs can be produced endogenously bydegradation of longer dsRNA molecules by an RNase III-related nucleasecalled Dicer. (Bernstein et al., Nature 409:363-366, 2001). siRNAs canalso be introduced into a cell exogenously, or by transcription of anexpression construct. Once formed, the siRNAs assemble with proteincomponents into endoribonuclease-containing complexes known asRNA-induced silencing complexes (RISCs). An ATP-generated unwinding ofthe siRNA activates the RISCs, which in turn target the complementarymRNA transcript by Watson-Crick base-pairing. Without wishing to bebound by any particular theory, it is believed that a RISC is guided toa target mRNA, where the siRNA duplex interacts sequence-specifically tomediate cleavage in a catalytic fashion (Bernstein et al., Nature409:363-366, 2001; Boutla et al., Curr Biol 11:1776-1780, 2001).Cleavage of the mRNA takes place near the middle of the region bound bythe siRNA strand. This sequence specific mRNA degradation results ingene silencing.

RNAi has been used to analyze gene function and to identify essentialgenes in mammalian cells (Elbashir et al., Methods 26:199-213, 2002;Harborth et al., J Cell Sci 114:4557-4565, 2001), including by way ofnon-limiting example neurons (Krichevsky et al., Proc Natl Acad Sci USA99:11926-11929, 2002). RNAi is also being evaluated for therapeuticmodalities, such as inhibiting or blocking the infection, replicationand/or growth of viruses, including without limitation poliovirus(Gitlin et al., Nature 418:379-380, 2002) and HIV (Capodici et al., JImmunol 169:5196-5201, 2002), and reducing expression of oncogenes(e.g., the bcr-abl gene; Scherr et al., Blood September 26 epub ahead ofprint, 2002). RNAi has been used to modulate gene expression inmammalian (mouse) and amphibian (Xenopus) embryos (respectively,Calegari et al., Proc Natl Acad Sci USA 99:14236-14240, 2002; and Zhou,et al., Nucleic Acids Res 30:1664-1669, 2002), and in postnatal mice(Lewis et al., Nat Genet. 32:107-108, 2002), and to reduce trangseneexpression in adult transgenic mice (McCaffrey et al., Nature 418:38-39,2002). Methods have been described for determining the efficacy andspecificity of siRNAs in cell culture and in vivo (see, e.g., Bertrandet al., Biochem Biophys Res Commun 296:1000-1004, 2002; Lassus et al.,Sci STKE 2002(147):PL13, 2002; and Leirdal et al., Biochem Biophys ResCommun 295:744-748, 2002).

Molecules that mediate RNAi, including without limitation siRNA, can beproduced in vitro by chemical synthesis (Hohjoh, FEBS Lett 521:195-199,2002), hydrolysis of dsRNA (Yang et al., Proc Natl Acad Sci USA99:9942-9947, 2002), by in vitro transcription with T7 RNA polymerase(Donzeet et al., Nucleic Acids Res 30:e46, 2002; Yu et al., Proc NatlAcad Sci USA 99:6047-6052, 2002), and by hydrolysis of double-strandedRNA using a nuclease such as E. coli RNase III (Yang et al., Proc NatlAcad Sci USA 99:9942-9947, 2002).

siRNA molecules may also be formed by annealing two oligonucleotides toeach other, typically have the following general structure, whichincludes both double-stranded and single-stranded portions:

                    |-m-| (Overhang)         |-----x----| (“Core”)     5′-XXXXXXXXXXXXNNNNN-3′ (SEQ ID NO:88)         ::::::::::::3′-NNNNNYYYYYYYYYYYY-5′ (SEQ ID NO:89)    |-n-| (Overhang)

Wherein N, X and Y are nucleotides; X hydrogen bonds to Y; “:” signifiesa hydrogen bond between two bases; x is a natural integer having a valuebetween 1 and about 100; and m and n are whole integers having,independently, values between 0 and about 100. In some embodiments, N, Xand Y are independently A, G, C and T or U. Non-naturally occurringbases and nucleotides can be present, particularly in the case ofsynthetic siRNA (i.e., the product of annealing two oligonucleotides).The double-stranded central section is called the “core” and has basepairs (bp) as units of measurement; the single-stranded portions areoverhangs, having nucleotides (nt) as units of measurement The overhangsshown are 3′ overhangs, but molecules with 5′ overhangs are also withinthe scope of the invention. Also within the scope of the invention aresiRNA molecules with no overhangs (i.e., m=0 and n=0), and those havingan overhang on one side of the core but not the other (e.g., m=0 andn>1, or vice-versa).

Initially, RNAi technology did not appear to be readily applicable tomammalian systems. This is because, in mammals, dsRNA activatesdsRNA-activated protein kinase (PKR) resulting in an apoptotic cascadeand cell death (Der et al., Proc. Natl. Acad. Sci. USA 94:3279-3283,1997). In addition, it has long been known that dsRNA activates theinterferon cascade in mammalian cells, which can also lead to alteredcell physiology (Colby et al., Annu. Rev. Microbiol. 25:333, 1971;Kleinschmidt et al., Annu. Rev. Biochem. 41:517, 1972; Lampson et al.,Proc. Natl. Acad. Sci. USA 58:L782, 1967; Lomniczi et al., J. Gen.Virol. 8:55, 1970; and Younger et al., J. Bacteriol. 92:862, 1966).However, dsRNA-mediated activation of the PKR and interferon cascadesrequires dsRNA longer than about 30 base pairs. In contrast, dsRNA lessthan 30 base pairs in length has been demonstrated to cause RNAi inmammalian cells (Caplen et al., Proc. Natl. Acad. Sci. USA 98:9742-9747,2001). Thus, it is expected that undesirable, non-specific effectsassociated with longer dsRNA molecules can be avoided by preparing shortRNA that is substantially free from longer dsRNAs.

References regarding siRNA: Bernstein et al., Nature 409:363-366, 2001;Boutla et al., Curr Biol 11:1776-1780, 2001; Cullen, Nat. Immunol.3:597-599, 2002; Caplen et al., Proc Natl Acad Sci USA 98:9742-9747,2001; Hamilton et al., Science 286:950-952, 1999; Nagase et al., DNARes. 6:63-70, 1999; Napoli et al., Plant Cell 2:279-289, 1990; Nicholsonet al., Mamm. Genome 13:67-73, 2002; Parrish et al., Mol Cell6:1077-1087, 2000; Romano et al., Mol Microbiol 6:3343-3353, 1992;Tabara et al., Cell 99:123-132, 1999; and Tuschl, Chembiochem.2:239-245, 2001.

Paddison et al. (Genes & Dev. 16:948-958, 2002) have used small RNAmolecules folded into hairpins as a means to effect RNAi. Accordingly,such short hairpin RNA (shRNA) molecules are also advantageously used inthe methods of the invention. The length of the stem and loop offunctional shRNAs varies; stem lengths can range anywhere from about 25to about 30 nt, and loop size can range between 4 to about 25 nt withoutaffecting silencing activity. While not wishing to be bound by anyparticular theory, it is believed that these shRNAs resemble the dsRNAproducts of the DICER RNase and, in any event, have the same capacityfor inhibiting expression of a specific gene.

In some embodiments, the invention provides that that siRNA or the shRNAinhibits NgR1 expression. In some embodiments, the invention furtherprovides that the siRNA or shRNA is at least 80%, 90%, or 95% identicalto the nucleotide sequence comprising: CUACUUCUCCCGCAGGCG (SEQ ID NO:52)or CCCGGACCGACGUCUUCAA (SEQ ID NO:54) or CUGACCACUGAGUCUUCCG (SEQ IDNO:56). In other embodiments, the siRNA or shRNA nucleotide sequence isCUACUUCUCCCGCAGGCG (SEQ ID NO:52) or CCCGGACCGACGUCUUCAA (SEQ ID NO:54)or CUGACCACUGAGUCUUCCG (SEQ ID NO:56).

In some embodiments, the invention further provides that the siRNA orshRNA nucleotide sequence is complementary to the mRNA produced by thepolynucleotide sequence GATGAAGAGGGCGTCC GCT (SEQ ID NO:53) orGGGCCTGGCTGCAGAAGTT (SEQ ID NO:55) or GACTGGTGACTCAGAG AAGGC (SEQ IDNO:57).

In some embodiments of the invention, the shRNA is expressed from alentiviral vector as described in Example 26.

Chemically synthesizing nucleic acid molecules with modifications (base,sugar and/or phosphate) can prevent their degradation by serumribonucleases, which can increase their potency (see e.g., Eckstein etal., International Publication No. WO 92/07065; Perrault et al., Nature344:565 (1990); Pieken et al., Science 253:314 (1991); Usman andCedergren, Trends in Biochem. Sci. 17:334 (1992); Usman et al.,International Publication No. WO 93/15187; and Rossi et al.,International Publication No. WO 91/03162; Sproat, U.S. Pat. No.5,334,711; Gold et al., U.S. Pat. No. 6,300,074; and Burgin et al.,supra; all of which are incorporated by reference herein). All of theabove references describe various chemical modifications that can bemade to the base, phosphate and/or sugar moieties of the nucleic acidmolecules described herein. Modifications that enhance their efficacy incells, and removal of bases from nucleic acid molecules to shortenoligonucleotide synthesis times and reduce chemical requirements aredesired.

There are several examples in the art describing sugar, base andphosphate modifications that can be introduced into nucleic acidmolecules with significant enhancement in their nuclease stability andefficacy. For example, oligonucleotides are modified to enhancestability and/or enhance biological activity by modification withnuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro,2′-O-methyl, 2′-O-allyl, 2′-H, nucleotide base modifications (for areview see Usman and Cedergren, TIBS. 17:34 (1992); Usman et al.,Nucleic Acids Symp. Ser. 31:163 (1994); Burgin et al., Biochemistry35:14090 (1996)). Sugar modification of nucleic acid molecules have beenextensively described in the art (see Eckstein et al., InternationalPublication PCT No. WO 92/07065; Perrault et al., Nature 344: 565-568(1990); Pieken et al., Science 253: 314-317 (1991); Usman and Cedergren,Trends in Biochem. Sci. 17: 334-339 (1992); Usman et al., InternationalPublication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 andBeigelman et al., J. Biol. Chem. 270:25702 (1995); Beigelman et al.,International PCT publication No. WO 97/26270; Beigelman et al., U.S.Pat. No. 5,716,824; Usman et al., U.S. Pat. No. 5,627,053; Woolf et al.,International PCT Publication No. WO 98/13526; Karpeisky et al., 1998,Tetrahedron Lett. 39:1131 (1998); Earnshaw and Gait, Biopolymers(Nucleic Acid Sciences) 48:39-55 (1998); Verma and Eckstein, Annu. Rev.Biochem. 67:99-134 (1998); and Burlina et al., Bioorg. Med. Chem.5:1999-2010 (1997); all of the references are hereby incorporated intheir totality by reference herein). Such publications describe generalmethods and strategies to determine the location of incorporation ofsugar, base and/or phosphate modifications and the like into nucleicacid molecules without modulating catalysis, and are incorporated byreference herein. In view of such teachings, similar modifications canbe used as described herein to modify the siRNA nucleic acid moleculesof the instant invention so long as the ability of siRNA to promote RNAiis cells is not significantly inhibited.

The invention features modified siRNA molecules, with phosphate backbonemodifications comprising one or more phosphorothioate,phosphorodithioate, methylphosphonate, phosphotriester, morpholino,amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate,sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl,substitutions. For a review of oligonucleotide backbone modifications,see Hunziker and Leumann, Nucleic Acid Analogues: Synthesis andProperties, in Modern Synthetic Methods, VCH, 331-417 (1995), andMesmaeker et al., Novel Backbone Replacements for Oligonucleotides, inCarbohydrate Modifications in Antisense Research, ACS, 24-39 (1994).

While chemical modification of oligonucleotide internucleotide linkageswith phosphorothioate, phosphorothioate, and/or 5′-methylphosphonatelinkages improves stability, excessive modifications can cause sometoxicity or decreased activity. Therefore, when designing nucleic acidmolecules, the amount of these internucleotide linkages should beminimized. The reduction in the concentration of these linkages shouldlower toxicity, resulting in increased efficacy and higher specificityof these molecules.

siRNA molecules having chemical modifications that maintain or enhanceactivity are provided. Such a nucleic acid is also generally moreresistant to nucleases than an unmodified nucleic acid. Accordingly, thein vitro and/or in vivo activity should not be significantly lowered. Incases in which modulation is the goal, therapeutic nucleic acidmolecules delivered exogenously should optimally be stable within cellsuntil translation of the target RNA has been modulated long enough toreduce the levels of the undesirable protein. This period of time variesbetween hours to days depending upon the disease state. Improvements inthe chemical synthesis of RNA and DNA (Wincott et al., Nucleic AcidsRes. 23:2677 (1995); Caruthers et al., Methods in Enzymology 211:3-19(1992) (incorporated by reference herein)) have expanded the ability tomodify nucleic acid molecules by introducing nucleotide modifications toenhance their nuclease stability, as described above.

Polynucleotides of the present invention can include one or more (e.g.about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clamp nucleotides. AG-clamp nucleotide is a modified cytosine analog wherein themodifications confer the ability to hydrogen bond both Watson-Crick andHoogsteen faces of a complementary guanine within a duplex, see, e.g.,Lin and Matteucci, J. Am. Chem. Soc. 120:8531-8532 (1998). A singleG-clamp analog substitution within an oligonucleotide can result insubstantially enhanced helical thermal stability and mismatchdiscrimination when hybridized to complementary oligonucleotides. Theinclusion of such nucleotides in polynucleotides of the inventionresults in both enhanced affinity and specificity to nucleic acidtargets, complementary sequences, or template strands. Polynucleotidesof the present invention can also include one or more (e.g., about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, or more) LNA “locked nucleic acid” nucleotidessuch as a 2′,4′-C mythylene bicyclo nucleotide (see, e.g., Wengel etal., International PCT Publication No. WO 00/66604 and WO 99/14226).

The present invention also features conjugates and/or complexes of siRNAmolecules of the invention. Such conjugates and/or complexes can be usedto facilitate delivery of siRNA molecules into a biological system, suchas a cell. The conjugates and complexes provided by the instantinvention can impart therapeutic activity by transferring therapeuticcompounds across cellular membranes, altering the pharmacokinetics,and/or modulating the localization of nucleic acid molecules of theinvention. The present invention encompasses the design and synthesis ofnovel conjugates and complexes for the delivery of molecules, including,but not limited to, small molecules, lipids, phospholipids, nucleosides,nucleotides, nucleic acids, antibodies, toxins, negatively chargedpolymers and other polymers, for example proteins, peptides, hormones,carbohydrates, polyethylene glycols, or polyamines, across cellularmembranes. In general, the transporters described are designed to beused either individually or as part of a multi-component system, with orwithout degradable linkers. These compounds are expected to improvedelivery and/or localization of nucleic acid molecules of the inventioninto a number of cell types originating from different tissues, in thepresence or absence of serum (see Sullenger and Cech, U.S. Pat. No.5,854,038). Conjugates of the molecules described herein can be attachedto biologically active molecules via linkers that are biodegradable,such as biodegradable nucleic acid linker molecules.

Therapeutic polynucleotides (e.g., siRNA molecules) deliveredexogenously optimally are stable within cells until reversetranscription of the RNA has been modulated long enough to reduce thelevels of the RNA transcript. The nucleic acid molecules are resistantto nucleases in order to function as effective intracellular therapeuticagents. Improvements in the chemical synthesis of nucleic acid moleculesdescribed in the instant invention and in the art have expanded theability to modify nucleic acid molecules by introducing nucleotidemodifications to enhance their nuclease stability as described above.

The present invention also provides for siRNA molecules having chemicalmodifications that maintain or enhance enzymatic activity of proteinsinvolved in RNAi. Such nucleic acids are also generally more resistantto nucleases than unmodified nucleic acids. Thus, in vitro and/or invivo the activity should not be significantly lowered.

Use of the polynucleotide-based molecules of the invention will lead tobetter treatment of the disease progression by affording the possibilityof combination therapies (e.g., multiple siRNA molecules targeted todifferent genes; nucleic acid molecules coupled with known smallmolecule modulators; or intermittent treatment with combinations ofmolecules, including different motifs and/or other chemical orbiological molecules). The treatment of subjects with siRNA moleculescan also include combinations of different types of nucleic acidmolecules, such as enzymatic nucleic acid molecules (ribozymes),allozymes, antisense, 2,5-A oligoadenylate, decoys, aptamers etc.

In another aspect, a siRNA molecule of the invention can comprise one ormore 5′ and/or a 3′-cap structures, for example on only the sense siRNAstrand, antisense siRNA strand, or both siRNA strands.

By “cap structure” is meant chemical modifications, which have beenincorporated at either terminus of the oligonucleotide (see, forexample, Adamic et al., U.S. Pat. No. 5,998,203, incorporated byreference herein). These terminal modifications protect the nucleic acidmolecule from exonuclease degradation, and may help in delivery and/orlocalization within a cell. The cap may be present at the 5′-terminus(5′-cap) or at the 3′-terminal (3′-cap) or may be present on bothtermini. In non-limiting examples: the 5′-cap is selected from the groupcomprising inverted abasic residue (moiety); 4′,5′-methylene nucleotide;1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide; carbocyclicnucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides;alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage;threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide,3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety;3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasic moiety;1,4-butanediol phosphate; 3′-phosphoramidate; hexylphosphate; aminohexylphosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; orbridging or non-bridging methylphosphonate moeity.

The 3′-cap can be selected from a group comprising, 4′,5′-methylenenucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide,carbocyclic nucleotide; 5′-amino-allyl phosphate; 1,3-diamino-2-propylphosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate;1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitolnucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide;phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seconucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentylnucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasicmoiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediolphosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate,phosphorothioate and/or phosphorodithioate, bridging or non bridgingmethylphosphonate and 5′-mercapto moieties (for more details seeBeaucage and Iyer, Tetrahedron 49:1925 (1993); incorporated by referenceherein).

Various modifications to nucleic acid siRNA structure can be made toenhance the utility of these molecules. Such modifications will enhanceshelf-life, half-life in vitro, stability, and ease of introduction ofsuch oligonucleotides to the target site, e.g. to enhance penetration ofcellular membranes, and confer the ability to recognize and bind totargeted cells.

Antisense technology can be used to control gene expression throughantisense DNA or RNA, or through triple-helix formation. Antisensetechniques are discussed for example, in Okano, J. Neurochem. 56:560(1991); Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, Fla. (1988). Triple helix formationis discussed in, for instance, Lee et al., Nucleic Acids Research 6:3073(1979); Cooney et al., Science 241:456 (1988); and Dervan et al.,Science 251:1300 (1991). The methods are based on binding of apolynucleotide to a complementary DNA or RNA.

For example, the 5′ coding portion of a polynucleotide that encodes NGR1may be used to design an antisense RNA oligonucleotide of from about 10to 40 base pairs in length. A DNA oligonucleotide is designed to becomplementary to a region of the gene involved in transcription therebypreventing transcription and the production of the target protein. Theantisense RNA oligonucleotide hybridizes to the mRNA in vivo and blockstranslation of the mRNA molecule into the target polypeptide.

In one embodiment, antisense nucleic acids specific for the NGR1 geneare produced intracellularly by transcription from an exogenoussequence. For example, a vector or a portion thereof, is transcribed,producing an antisense nucleic acid (RNA). Such a vector can remainepisomal or become chromosomally integrated, as long as it can betranscribed to produce the desired antisense RNA. Such vectors can beconstructed by recombinant DNA technology methods standard in the art.Vectors can be plasmid, viral, or others known in the art, used forreplication and expression in vertebrate cells. Expression of theantisense molecule, can be by any promoter known in the art to act invertebrate, preferably human cells, such as those described elsewhereherein.

Absolute complementarity of an antisense molecule, although preferred,is not required. A sequence complementary to at least a portion of anRNA encoding NgR1, means a sequence having sufficient complementarity tobe able to hybridize with the RNA, forming a stable duplex; or triplexformation may be assayed. The ability to hybridize will depend on boththe degree of complementarity and the length of the antisense nucleicacid. Generally, the larger the hybridizing nucleic acid, the more basemismatches it may contain and still form a stable duplex (or triplex asthe case may be). One skilled in the art can ascertain a tolerabledegree of mismatch by use of standard procedures to determine themelting point of the hybridized complex.

Oligonucleotides that are complementary to the 5′ end of a messengerRNA, e.g., the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs have been shown to be effective at inhibitingtranslation of mRNAs as well. See generally, Wagner, R., Nature372:333-335 (1994). Thus, oligonucleotides complementary to either the5′- or 3′-non-translated, non-coding regions could be used in anantisense approach to inhibit translation of NgR1. Oligonucleotidescomplementary to the 5′ untranslated region of the mRNA should includethe complement of the AUG start codon. Antisense oligonucleotidescomplementary to mRNA coding regions are less efficient inhibitors oftranslation but could be used in accordance with the invention.Antisense nucleic acids should be at least six nucleotides in length,and are preferably oligonucleotides ranging from 6 to about 50nucleotides in length. In specific aspects the oligonucleotide is atleast 10 nucleotides, at least 17 nucleotides, at least 25 nucleotidesor at least 50 nucleotides.

Polynucleotides for use in the therapeutic methods disclosed herein,including aptamers described below, can be DNA or RNA or chimericmixtures or derivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al., Proc. Natl. Acad. Sci. U.S.A.86:6553-6556 (1989); Lemaitre et al., Proc. Natl. Acad. Sci. 84:648-652(1987)); PCT Publication No. WO88/09810, published Dec. 15, 1988) or theblood-brain barrier (see, e.g., PCT Publication No. WO89/10134,published Apr. 25, 1988), hybridization-triggered cleavage agents. (See,e.g., Krol et al., BioTechniques 6:958-976 (1988)) or intercalatingagents. (See, e.g., Zon, Pharm. Res. 5:539-549 (1988)). To this end, theoligonucleotide may be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

An antisense oligonucleotide for use in the therapeutic methodsdisclosed herein may comprise at least one modified base moiety which isselected from the group including, but not limited to, 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine,4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N-6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N-6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

An antisense oligonucleotide for use in the therapeutic methodsdisclosed herein may also comprise at least one modified sugar moietyselected from the group including, but not limited to, arabinose,2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, an antisense oligonucleotide for use in thetherapeutic methods disclosed herein comprises at least one modifiedphosphate backbone selected from the group including, but not limitedto, a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, aphosphoramidate, a phosphordiamidate, a methylphosphonate, an alkylphosphotriester, and a formacetal or analog thereof.

In yet another embodiment, an antisense oligonucleotide for use in thetherapeutic methods disclosed herein is an α-anomeric oligonucleotide.An α-anomeric oligonucleotide forms specific double-stranded hybridswith complementary RNA in which, contrary to the usual situation, thestrands run parallel to each other (Gautier et al., Nucl. Acids Res.15:6625-6641 (1987)). The oligonucleotide is a 2′-0-methylribonucleotide(Inoue et al., Nucl. Acids Res. 15:6131-6148 (1987)), or a chimericRNA-DNA analogue (Inoue et al., FEBS Lett. 215:327-330 (1987)).

Polynucleotides of the invention, including aptamers may be synthesizedby standard methods known in the art, e.g. by use of an automated DNAsynthesizer (such as are commercially available from Biosearch, AppliedBiosystems, etc.). As examples, phosphorothioate oligonucleotides may besynthesized by the method of Stein et al., Nucl. Acids Res. 16:3209(1988), methylphosphonate oligonucleotides can be prepared by use ofcontrolled pore glass polymer supports (Sarin et al., Proc. Natl. Acad.Sci. U.S.A. 85:7448-7451 (1988)), etc.

Polynucleotide compositions for use in the therapeutic methods disclosedherein further include catalytic RNA, or a ribozyme (See, e.g., PCTInternational Publication WO 90/11364, published Oct. 4, 1990; Sarver etal, Science 247:1222-1225 (1990). The use of hammerhead ribozymes ispreferred. Hammerhead ribozymes cleave mRNAs at locations dictated byflanking regions that form complementary base pairs with the targetmRNA. The sole requirement is that the target mRNA have the followingsequence of two bases: 5′-UG-3′. The construction and production ofhammerhead ribozymes is well known in the art and is described morefully in Haseloff and Gerlach, Nature 334:585-591 (1988). Preferably,the ribozyme is engineered so that the cleavage recognition site islocated near the 5′ end of the target mRNA; i.e., to increase efficiencyand minimize the intracellular accumulation of non-functional mRNAtranscripts.

As in the antisense approach, ribozymes for use in the diagnostic andtherapeutic methods disclosed herein can be composed of modifiedoligonucleotides (e.g. for improved stability, targeting, etc.) and maybe delivered to cells which express NgR1 in vivo. DNA constructsencoding the ribozyme may be introduced into the cell in the same manneras described above for the introduction of antisense encoding DNA. Apreferred method of delivery involves using a DNA construct “encoding”the ribozyme under the control of a strong constitutive promoter, suchas, for example, pol III or pol II promoter, so that transfected cellswill produce sufficient quantities of the ribozyme to destroy endogenousNgR1 messages and inhibit translation. Since ribozymes unlike antisensemolecules, are catalytic, a lower intracellular concentration isrequired for efficiency.

Aptamers

In another embodiment, the NgR1 antagonist for use in the methods of thepresent invention is an aptamer. An aptamer can be a nucleotide or apolypeptide which has a unique sequence, has the property of bindingspecifically to a desired target (e.g. a polypeptide), and is a specificligand of a given target. Nucleotide aptamers of the invention includedouble stranded DNA and single stranded RNA molecules that bind to NgR1.

Nucleic acid aptamers are selected using methods known in the art, forexample via the Systematic Evolution of Ligands by ExponentialEnrichment (SELEX) process. SELEX is a method for the in vitro evolutionof nucleic acid molecules with highly specific binding to targetmolecules as described in e.g. U.S. Pat. Nos. 5,475,096, 5,580,737,5,567,588, 5,707,796, 5,763,177, 6,011,577, and 6,699,843, incorporatedherein by reference in their entirety. Another screening method toidentify aptamers is described in U.S. Pat. No. 5,270,163 (alsoincorporated herein by reference). The SELEX process is based on thecapacity of nucleic acids for forming a variety of two- andthree-dimensional structures, as well as the chemical versatilityavailable within the nucleotide monomers to act as ligands (formspecific binding pairs) with virtually any chemical compound, whethermonomeric or polymeric, including other nucleic acid molecules andpolypeptides. Molecules of any size or composition can serve as targets.

The SELEX method involves selection from a mixture of candidateoligonucleotides and step-wise iterations of binding, partitioning andamplification, using the same general selection scheme, to achievedesired binding affinity and selectivity. Starting from a mixture ofnucleic acids, preferably comprising a segment of randomized sequence,the SELEX method includes steps of contacting the mixture with thetarget under conditions favorable for binding; partitioning unboundnucleic acids from those nucleic acids which have bound specifically totarget molecules; dissociating the nucleic acid-target complexes;amplifying the nucleic acids dissociated from the nucleic acid-targetcomplexes to yield a ligand enriched mixture of nucleic acids. The stepsof binding, partitioning, dissociating and amplifying are repeatedthrough as many cycles as desired to yield highly specific high affinitynucleic acid ligands to the target molecule.

Nucleotide aptamers may be used, for example, as diagnostic tools or asspecific inhibitors to dissect intracellular signaling and transportpathways (James (2001) Curr. Opin. Pharmacol. 1:540-546). The highaffinity and specificity of nucleotide aptamers makes them goodcandidates for drug discovery. For example, aptamer antagonists to thetoxin ricin have been isolated and have IC50 values in the nanomolarrange (Hesselberth J R et al. (2000) J Biol Chem 275:4937-4942).Nucleotide aptamers may also be used against infectious disease,malignancy and viral surface proteins to reduce cellular infectivity.

Nucleotide aptamers for use in the methods of the present invention maybe modified (e.g., by modifying the backbone or bases or conjugated topeptides) as described herein for other polynucleotides.

Using the protein structure of NgR1, screening for aptamers that act onNgR1 using the SELEX process would allow for the identification ofaptamers that inhibit NgR1-mediated processes (e.g., NgR1-mediatedinhibition of axonal regeneration).

Polypeptide aptamers for use in the methods of the present invention arerandom peptides selected for their ability to bind to and thereby blockthe action of NgR1. Polypeptide aptamers may include a short variablepeptide domain attached at both ends to a protein scaffold. This doublestructural constraint greatly increases the binding affinity of thepeptide aptamer to levels comparable to an antibody's (nanomolar range).See, e.g., Hoppe-Seyler F et al. (2000) J Mol Med 78(8):426-430. Thelength of the short variable peptide is typically about 10 to 20 aminoacids, and the scaffold may be any protein which has good solubility andcompacity properties. One non-limiting example of a scaffold protein isthe bacterial protein Thioredoxin-A. See, e.g., Cohen B A et al. (1998)PNAS 95(24): 14272-14277.

Polypeptide aptamers are peptides or small polypeptides that act asdominant inhibitors of protein function. Peptide aptamers specificallybind to target proteins, blocking their functional ability (Kolonin etal. (1998) Proc. Natl. Acad. Sci. 95: 14,266-14,271). Peptide aptamersthat bind with high affinity and specificity to a target protein can beisolated by a variety of techniques known in the art. Peptide aptamerscan be isolated from random peptide libraries by yeast two-hybridscreens (Xu, C. W., et al. (1997) Proc. Natl. Acad. Sci.94:12,473-12,478) or by ribosome display (Hanes et al. (1997) Proc.Natl. Acad. Sci. 94:4937-4942). They can also be isolated from phagelibraries (Hoogenboom, H. R., et al. (1998) Immunotechnology 4:1-20) orchemically generated peptide libraries. Additionally, polypeptideaptamers may be selected using the selection of Ligand Regulated PeptideAptamers (LiRPAs). See, e.g., Binkowski B F et al., (2005) Chem & Biol12(7): 847-855, incorporated herein by reference. Although the difficultmeans by which peptide aptamers are synthesized makes their use morecomplex than polynucleotide aptamers, they have unlimited chemicaldiversity. Polynucleotide aptamers are limited because they utilize onlythe four nucleotide bases, while peptide aptamers would have amuch-expanded repertoire (i.e., 20 amino acids).

Peptide aptamers for use in the methods of the present invention may bemodified (e.g., conjugated to polymers or fused to proteins) asdescribed for other polypeptides elsewhere herein.

Compositions

In some embodiments, the invention provides compositions comprising apolypeptide selected from the group consisting of SEQ ID NOs: 1-5,26-27, 29-37 and 41-45.

In some embodiments, the invention provides compositions comprising ananti-Nogo receptor-1 antibody or an antigen-binding fragment thereof, ora soluble Nogo receptor-1 polypeptide or fusion protein of the presentinvention.

In some embodiments, the invention provides a composition comprising apolynucleotide of the present invention.

In some embodiments, the invention provides compositions comprising apolypeptide of the present invention and an anti-inflammatory agent.

In some embodiments, the present invention may contain suitablepharmaceutically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically for delivery to the siteof action. Suitable formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form, forexample, water-soluble salts. In addition, suspensions of the activecompounds as appropriate oily injection suspensions may be administered.Suitable lipophilic solvents or vehicles include fatty oils, forexample, sesame oil, or synthetic fatty acid esters, for example, ethyloleate or triglycerides. Aqueous injection suspensions may containsubstances which increase the viscosity of the suspension include, forexample, sodium carboxymethyl cellulose, sorbitol and dextran.Optionally, the suspension may also contain stabilizers. Liposomes canalso be used to encapsulate the molecules of this invention for deliveryinto the cell. Exemplary “pharmaceutically acceptable carriers” are anyand all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and the likethat are physiologically compatible, water, saline, phosphate bufferedsaline, dextrose, glycerol, ethanol and the like, as well ascombinations thereof. In some embodiments, the composition comprisesisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride. In some embodiments, the compositionscomprise pharmaceutically acceptable substances such as wetting or minoramounts of auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the antibodies, antigen-binding fragments, soluble Nogo receptors orfusion proteins of the invention.

Compositions of the invention may be in a variety of forms, including,for example, liquid, semi-solid and solid dosage forms, such as liquidsolutions (e.g., injectable and infusible solutions), dispersions orsuspensions. The preferred form depends on the intended mode ofadministration and therapeutic application. In one embodiment,compositions are in the form of injectable or infusible solutions, suchas compositions similar to those used for passive immunization of humanswith other antibodies.

The composition can be formulated as a solution, micro emulsion,dispersion, liposome, or other ordered structure suitable to high drugconcentration. Sterile injectable solutions can be prepared byincorporating an anti-Nogo receptor-1 antibody in the required amount inan appropriate solvent with one or a combination of ingredientsenumerated above, as required, followed by filtered sterilization.Generally, dispersions are prepared by incorporating the active compoundinto a sterile vehicle that contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The proper fluidity of a solution can be maintained, for example, by theuse of a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

In some embodiments, the active compound may be prepared with a carrierthat will protect the compound against rapid release, such as acontrolled release formulation, including implants, transdermal patches,and microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York (1978).

Supplementary active compounds also can be incorporated into thecompositions. In some embodiments, a Nogo receptor-1 antibody or anantigen-binding fragments thereof, or soluble Nogo receptor-1polypeptides or fusion proteins of the invention are coformulated withand/or coadministered with one or more additional therapeutic agents,including, for example, an anti-inflammatory agent. In one embodiment,the anti-inflammatory agent is a non-steroidal anti-inflammatory agent.In another embodiment, the anti-inflammatory agent is a steroidalanti-inflammatory agent. In a particular embodiment, theanti-inflammatory agent is methylprednisolone.

In one embodiment, the present invention is directed to the use of aNogo receptor antagonist in combination with a non-steroidalanti-inflammatory agent (NSAID), prodrug esters or pharmaceuticallyacceptable salts thereof. Examples of NSAIDs which are well-known in theart include propionic acid derivatives (e.g., alminoprofen,benoxaprofen, bucloxic acid, carprofen, fenbufen, fenoprofen, fluprofen,flurbiprofen, ibuprofen, indoprofen, ketoprofen, miroprofen, naproxen,oxaprozin, pirprofen, pranoprofen, suprofen, tiaprofenic acid andtioxaprofen), acetic acid derivatives (e.g., indomethacin, acemetacin,alclofenac, clidanac, diclofenac, fenclofenac, fenclozic acid,fentiazac, furofenac, ibufenac, isoxepac, oxpinac, sulindac, tiopinac,tolmetin, zidometacin and zomepirac), fenamic acid derivatives (e.g.,flufenamic acid, meclofenamic acid, mefenamic acid, niflumic acid andtolfenamic acid), biphenylcarboxylic acid derivatives (e.g., diflunisaland flufenisal), oxicams (e.g., isoxicam, piroxicam, sudoxicam andtenoxicam), salicylates (e.g., acetyl salicylic acid and sulfasalazine)and the pyrazolones (e.g., apazone, bezpiperylon, feprazone,mofebutazone, oxyphenbutazone and phenylbutazone); Structurally relatedNSAIDs having similar analgesic and anti-inflammatory properties to theNSAIDs are also intended to be encompassed by this group.

In another embodiment, the present invention is directed to the use of aNogo receptor antagonist in combination with any of one or moresteroidal anti-inflammatory agents such as corticosteroids, prodrugesters or pharmaceutically acceptable salts thereof. Non-limitingexamples of such steroidal agents include hydrocortisone and compoundswhich are derived from hydrocortisone, such as 21-acetoxypregnenolone,alclomerasone, algestone, amcinonide, beclomethasone, betamethasone,betamethasone valerate, budesonide, chloroprednisone, clobetasol,clobetasol propionate, clobetasone, clobetasone butyrate, clocortolone,cloprednol, corticosterone, cortisone, cortivazol, deflazacon, desonide,desoximerasone, dexamethasone, diflorasone, diflucortolone,difluprednate, enoxolone, fluazacort, flucloronide, flumethasone,flumethasone pivalate, flunisolide, flucinolone acetonide, fluocinonide,fluorocinolone acetonide, fluocortin butyl, fluocortolone,fluorocortolone hexanoate, diflucortolone valerate, fluorometholone,fluperolone acetate, fluprednidene acetate, fluprednisolone,flurandenolide, formocortal, halcinonide, halometasone, halopredoneacetate, hydrocortamate, hydrocortisone, hydrocortisone acetate,hydrocortisone butyrate, hydrocortisone phosphate, hydrocortisone21-sodium succinate, hydrocortisone tebutate, mazipredone, medrysone,meprednisone, methylprednisolone, mometasone furoate, paramethasone,prednicarbate, prednisolone, prednisolone 21-diedryaminoacetate,prednisolone sodium phosphate, prednisolone sodium succinate,prednisolone sodium 21-m-sulfobenzoate, prednisolone sodium21-stearoglycolate, prednisolone tebutate, prednisolone21-trimethylacetate, prednisone, prednival, prednylidene, prednylidene21-diethylaminoacetate, tixocortol, triamcinolone, triamcinoloneacetonide, triamcinolone benetonide and triamcinolone hexacetonide.Structurally related corticosteroids having similar analgesic andanti-inflammatory properties are also intended to be encompassed by thisgroup. In one particular embodiment, the Nogo receptor antagonist isused in combination with methylprednisolone.

The pharmaceutical compositions of the invention may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of an antibody, antigen-binding fragment, polypeptide(s), orfusion protein of the invention. A “therapeutically effective amount”refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic result. A therapeuticallyeffective amount of the Nogo receptor-1 antibody or antigen-bindingfragment thereof, soluble Nogo receptor-1 polypeptide or Nogo receptorfusion protein may vary according to factors such as the disease state,age, sex, and weight of the individual. A therapeutically effectiveamount is also one in which any toxic or detrimental effects of theantibody, antigen-binding fragment, soluble Nogo receptor-1 polypeptideor Nogo receptor fusion protein are outweighed by the therapeuticallybeneficial effects. A “prophylactically effective amount” refers to anamount effective, at dosages and for periods of time necessary, toachieve the desired prophylactic result. Typically, since a prophylacticdose is used in subjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage unit formas used herein refers to physically discrete units suited as unitarydosages for the mammalian subjects to be treated, each unit containing apredetermined quantity of active compound calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the antibody, antigen-binding fragment, and solublereceptor-1 polypeptide or Nogo receptor fusion protein and theparticular therapeutic or prophylactic effect to be achieved, and (b)the limitations inherent in the art of compounding such an antibody,antigen-binding fragment, and soluble receptor-1 polypeptide or Nogoreceptor fusion protein for the treatment of sensitivity in individuals.In some embodiments a therapeutically effective dose range for Nogoreceptor-1 antibodies or antigen-binding fragments thereof is 0.1-4mg/Kg per day. In some embodiments a therapeutically effective doserange for Nogo receptor-1 antibodies or antigen-binding fragmentsthereof is 0.2-4 mg/Kg per day. In some embodiments a therapeuticallyeffective dose range for Nogo receptor-1 antibodies or antigen-bindingfragments thereof is 0.2 mg/Kg per day.

In the methods of the invention the NgR1 antagonists are generallyadministered directly to the nervous system, intracerebroventricularly,or intrathecally, e.g. into a chronic lesion of MS. Compositions foradministration according to the methods of the invention can beformulated so that a dosage of 0.001-10 mg/kg body weight per day of theNGR1 antagonist is administered. In some embodiments of the invention,the dosage is 0.01-1.0 mg/kg body weight per day. In some embodiments,the dosage is 0.001-0.5 mg/kg body weight per day.

For treatment with an NgR1 antagonist of the invention, the dosage canrange, e.g., from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5mg/kg (e.g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2mg/kg, etc.), of the host body weight. For example dosages can be 1mg/kg body weight or 10 mg/kg body weight or within the range of 1-10mg/kg, preferably at least 1 mg/kg. Doses intermediate in the aboveranges are also intended to be within the scope of the invention.Subjects can be administered such doses daily, on alternative days,weekly or according to any other schedule determined by empiricalanalysis. An exemplary treatment entails administration in multipledosages over a prolonged period, for example, of at least six months.Additional exemplary treatment regimes entail administration once perevery two weeks or once a month or once every 3 to 6 months. Exemplarydosage schedules include 1-10 mg/kg or 15 mg/kg on consecutive days, 30mg/kg on alternate days or 60 mg/kg weekly.

In some methods, two or more NgR1 antagonists are administeredsimultaneously, in which case the dosage of each antagonist administeredfalls within the ranges indicated. Supplementary active compounds alsocan be incorporated into the compositions used in the methods of theinvention. For example, an NgR1 antagonist may be coformulated withand/or coadministered with one or more additional therapeutic agents,such as an anti-inflammatory agent, for example, methylprednisolone.

The invention encompasses any suitable delivery method for an NgR1antagonist to a selected target tissue, including bolus injection of anaqueous solution or implantation of a controlled-release system. Use ofa controlled-release implant reduces the need for repeat injections.

The NgR1 antagonists used in the methods of the invention may bedirectly infused into the brain. Various implants for direct braininfusion of compounds are known and are effective in the delivery oftherapeutic compounds to human patients suffering from neurologicaldisorders. These include chronic infusion into the brain using a pump,stereotactically implanted, temporary interstitial catheters, permanentintracranial catheter implants, and surgically implanted biodegradableimplants. See, e.g., Gill et al., supra; Scharfen et al., “High ActivityIodine-125 Interstitial Implant For Gliomas,” Int. J. Radiation OncologyBiol. Phys. 24(4):583-91 (1992); Gaspar et al., “Permanent 125I Implantsfor Recurrent Malignant Gliomas,” Int. J. Radiation Oncology Biol. Phys.43(5):977-82 (1999); chapter 66, pages 577-580, Bellezza et al.,“Stereotactic Interstitial Brachytherapy,” in Gildenberg et al.,Textbook of Stereotactic and Functional Neurosurgery, McGraw-Hill(1998); and Brem et al., “The Safety of Interstitial Chemotherapy withBCNU-Loaded Polymer Followed by Radiation Therapy in the Treatment ofNewly Diagnosed Malignant Gliomas: Phase I Trial,” J. Neuro-Oncology26:111-23 (1995).

The compositions may also comprise an NGR1 antagonist of the inventiondispersed in a biocompatible carrier material that functions as asuitable delivery or support system for the compounds. Suitable examplesof sustained release carriers include semipermeable polymer matrices inthe form of shaped articles such as suppositories or capsules.Implantable or microcapsular sustained release matrices includepolylactides (U.S. Pat. No. 3,773,319; EP 58,481), copolymers ofL-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., Biopolymers22:547-56 (1985)); poly(2-hydroxyethyl-methacrylate), ethylene vinylacetate (Langer et al., J. Biomed. Mater. Res. 15:167-277 (1981);Langer, Chem. Tech. 12:98-105 (1982)) or poly-D-(−)-3hydroxybutyric acid(EP 133,988).

In some embodiments, an NGR1 antagonist of the invention is administeredto a patient by direct infusion into an appropriate region of the brain.See, e.g., Gill et al., “Direct brain infusion of glial cellline-derived neurotrophic factor in Parkinson disease,” Nature Med. 9:589-95 (2003). Alternative techniques are available and may be appliedto administer an NgR antagonist according to the invention. For example,stereotactic placement of a catheter or implant can be accomplishedusing the Riechert-Mundinger unit and the ZD (Zamorano-Dujovny)multipurpose localizing unit. A contrast-enhanced computerizedtomography (CT) scan, injecting 120 ml of omnipaque, 350 mg iodine/ml,with 2 mm slice thickness can allow three-dimensional multiplanartreatment planning (STP, Fischer, Freiburg, Germany). This equipmentpermits planning on the basis of magnetic resonance imaging studies,merging the CT and MRI target information for clear target confirmation.

The Leksell stereotactic system (Downs Surgical, Inc., Decatur, Ga.)modified for use with a GE CT scanner (General Electric Company,Milwaukee, Wis.) as well as the Brown-Roberts-Wells (BRW) stereotacticsystem (Radionics, Burlington, Mass.) can be used for this purpose.Thus, on the morning of the implant, the annular base ring of the BRWstereotactic frame can be attached to the patient's skull. Serial CTsections can be obtained at 3 mm intervals though the (target tissue)region with a graphite rod localizer frame clamped to the base plate. Acomputerized treatment planning program can be run on a VAX 11/780computer (Digital Equipment Corporation, Maynard, Mass.) using CTcoordinates of the graphite rod images to map between CT space and BRWspace.

Uses of the Antibodies, Antigen-Binding Fragments, Soluble Receptors,Fusion Proteins, Polynucleotides and Compositions

In some embodiments, the invention provides methods for inhibiting Nogoreceptor-1 activity by administering anti-Nogo receptor-1 antibodies,antigen-binding fragments of such antibodies, soluble Nogo receptor-1polypeptides, or fusion proteins comprising such polypeptides to amammal in need thereof.

In some embodiments, the invention provides a method of inhibiting Nogoreceptor-1 binding to a ligand, comprising the step of contacting Nogoreceptor-1 with an antibody or antigen-binding fragment of thisinvention. In some embodiments, the ligand is selected from the groupconsisting of NogoA, NogoB, NogoC, MAG and OM-gp.

In some embodiments, the invention provides a method for inhibitinggrowth cone collapse in a neuron, comprising the step of contacting theneuron with the antibody or antigen-binding fragment thereof of thisinvention. In some embodiments, the invention provides a method fordecreasing the inhibition of neurite outgrowth or sprouting in a neuron,comprising the step of contacting the neuron with the antibody orantigen-binding fragment of this invention. In some embodiments, theneuron is a CNS neuron. In some of these methods, the neurite outgrowthor sprouting is axonal growth.

In some embodiments, the invention provides a method of promotingsurvival of a neuron in a mammal, which neuron is at risk of dying,comprising (a) providing a cultured host cell expressing (i) ananti-Nogo receptor-1 antibody or antigen-binding fragment thereof; or(ii) a soluble Nogo receptor-1 polypeptide; and (b) introducing the hostcell into the mammal at or near the site of the neuron. AlmudenaRamon-Cueto, M Isabel Cordero, Fernando F Santos-Benito and Jesus Avila(2000) Functional recovery of paralegic rats and motor axon regenerationin their spinal cords by olfactory ensheathing cells. Neuron 25,425-435.

In some embodiments, the invention provides a gene therapy method ofpromoting survival of a neuron at risk of dying, which neuron is in amammal, comprising administering at or near the site of the neuron aviral vector comprising a nucleotide sequence that encodes (a) ananti-Nogo receptor-1 antibody or antigen-binding fragment thereof; or(b) a soluble Nogo receptor-1 polypeptide, wherein the anti-Nogoreceptor-1 antibody, antigen-binding fragment, or soluble Nogoreceptor-1 polypeptide is expressed from the nucleotide sequence in themammal in an amount sufficient to promote survival of the neuron. Viralvectors and methods useful for these embodiments are described in, e.g.,Noel et al., Human Gene Therapy, 13:1483-93 (2002).

In some embodiments, the invention provides a method of inhibiting Nogoreceptor-1 binding to a ligand, comprising the step of contacting theligand with the soluble Nogo receptor-1 polypeptide or the Nogoreceptor-1 fusion protein of this invention.

In some embodiments, the invention provides a method of modulating anactivity of a Nogo receptor-1 ligand, comprising the step of contactingthe Nogo receptor-1 ligand with a soluble Nogo receptor-1 polypeptide ora Nogo receptor-1 fusion protein of the invention.

In some embodiments, the invention provides a method for inhibitinggrowth cone collapse in a neuron, comprising the step of contacting aNogo receptor-1 ligand with a soluble Nogo receptor-1 polypeptide or aNogo receptor-1 fusion protein of this invention. In some embodiments,the invention provides a method for decreasing the inhibition of neuriteoutgrowth or sprouting in a neuron, comprising the step of contacting aNogo receptor-1 ligand with the soluble Nogo receptor-1 polypeptide orthe Nogo receptor-1 fusion protein of this invention. In someembodiments, the neuron is a CNS neuron. In some embodiments, the ligandis selected from the group consisting of NogoA, NogoB, NogoC, MAG andOM-gp. In some embodiments, the neurite outgrowth or sprouting is axonalgrowth.

In some embodiments, the invention provides a method for promotingneurite outgrowth comprising contacting a neuron with a polypeptide, apolynucleotide, or a composition of the invention. In some embodiments,the polypeptide, polynucleotide or composition inhibits neuriteoutgrowth inhibition. In some embodiments, the neuron is in a mammal. Insome embodiments, the mammal is a human.

In some embodiments, the invention provides a method of inhibitingsignal transduction by the NgR1 signaling complex, comprising contactinga neuron with an effective amount of a polypeptide, a polynucleotide, ora composition of the invention. In some embodiments, the neuron is in amammal. In some embodiments, the mammal is a human.

In some embodiments, the invention provides a method of treating acentral nervous system (CNS) disease, disorder, or injury in a mammal,comprising administering to a mammal in need of treatment an effectiveamount of a polypeptide, a polynucleotide, or a composition of thepresent invention. In some embodiments, the disease, disorder, or injuryis multiple sclerosis, ALS, Huntington's disease, Alzheimer's disease,Parkinson's disease, diabetic neuropathy, stroke, traumatic braininjuries, spinal cord injury, optic neuritis, glaucoma, hearing loss,and adrenal leukodystrophy.

Any of the types of antibodies or receptors described herein may be usedtherapeutically. In some embodiments, the anti-Nogo receptor-1 antibodyis a human antibody. In some embodiments, the mammal is a human patient.In some embodiments, the antibody or antigen-binding fragment thereof isadministered to a non-human mammal expressing a Nogo receptor-1 withwhich the antibody cross-reacts (e.g., a primate, cynomologous or rhesusmonkey) for veterinary purposes or as an animal model of human disease.Such animal models may be useful for evaluating the therapeutic efficacyof antibodies of this invention.

In some embodiments, administration of anti-Nogo receptor-1 antibody orantigen-binding fragment, or soluble Nogo receptor-1 polypeptide orfusion protein is used to treat a spinal cord injury to facilitateaxonal growth throughout the injured site.

The anti-Nogo receptor-1 antibodies or antigen-binding fragments, orsoluble Nogo receptor-1 polypeptides or fusion proteins of the presentinvention can be provided alone, or in combination, or in sequentialcombination with other agents that modulate a particular pathologicalprocess. For example, anti-inflammatory agents may be co-administeredfollowing stroke as a means for blocking further neuronal damage andinhibition of axonal regeneration. As used herein, the Nogo receptor-1antibodies, antigen-binding fragments, soluble Nogo receptor-1 and Nogoreceptor fusion proteins, are said to be administered in combinationwith one or more additional therapeutic agents when the two areadministered simultaneously, consecutively or independently.

The anti-Nogo receptor-1 antibodies, antigen-binding fragments, solubleNogo receptor-1 polypeptides, Nogo receptor-1 fusion proteins of thepresent invention can be administered via parenteral, subcutaneous,intravenous, intramuscular, intraperitoneal, transdermal, inhalationalor buccal routes. For example, an agent may be administered locally to asite of injury via microinfusion. Typical sites include, but are notlimited to, damaged areas of the spinal cord resulting from injury. Thedosage administered will be dependent upon the age, health, and weightof the recipient, kind of concurrent treatment, if any, frequency oftreatment, and the nature of the effect desired.

The compounds of this invention can be utilized in vivo, ordinarily inmammals, such as humans, sheep, horses, cattle, pigs, dogs, cats, ratsand mice, or in vitro.

Vectors of the Invention

In some embodiments, the invention provides recombinant DNA molecules(rDNA) that contain a coding sequence. As used herein, a rDNA moleculeis a DNA molecule that has been subjected to molecular manipulation.Methods for generating rDNA molecules are well known in the art, forexample, see Sambrook et al., Molecular Cloning—A Laboratory Manual,Cold Spring Harbor Laboratory Press (1989). In some rDNA molecules, acoding DNA sequence is operably linked to expression control sequencesand vector sequences.

In some embodiments, the invention provides vectors comprising thenucleic acids encoding the polypeptides of the invention. The choice ofvector and expression control sequences to which the nucleic acids ofthis invention is operably linked depends directly, as is well known inthe art, on the functional properties desired (e.g., protein expression,and the host cell to be transformed). A vector of the present inventionmay be at least capable of directing the replication or insertion intothe host chromosome, and preferably also expression, of the structuralgene included in the rDNA molecule.

Expression control elements that are used for regulating the expressionof an operably linked protein encoding sequence are known in the art andinclude, but are not limited to, inducible promoters, constitutivepromoters, secretion signals, and other regulatory elements. Preferably,the inducible promoter is readily controlled, such as being responsiveto a nutrient in the host cell's medium.

In one embodiment, the vector containing a coding nucleic acid moleculewill include a prokaryotic replicon, i.e., a DNA sequence having theability to direct autonomous replication and maintenance of therecombinant DNA molecule extra-chromosomally in a prokaryotic host cell,such as a bacterial host cell, transformed therewith. Such replicons arewell known in the art. In addition, vectors that include a prokaryoticreplicon may also include a gene whose expression confers a detectableor selectable marker such as a drug resistance. Typical of bacterialdrug resistance genes are those that confer resistance to ampicillin ortetracycline.

Vectors that include a prokaryotic replicon can further include aprokaryotic or bacteriophage promoter capable of directing theexpression (transcription and translation) of the coding gene sequencesin a bacterial host cell, such as E. coli. A promoter is an expressioncontrol element formed by a DNA sequence that permits binding of RNApolymerase and transcription to occur. Promoter sequences compatiblewith bacterial hosts are typically provided in plasmid vectorscontaining convenient restriction sites for insertion of a DNA segmentof the present invention. Examples of such vector plasmids are pUC8,pUC9, pBR322 and pBR329 (Bio-Rad® Laboratories), pPL and pKK223(Pharmacia). Any suitable prokaryotic host can be used to express arecombinant DNA molecule encoding a protein of the invention.

Expression vectors compatible with eukaryotic cells, preferably thosecompatible with vertebrate cells, can also be used to form a rDNAmolecules that contains a coding sequence. Eukaryotic cell expressionvectors are well known in the art and are available from severalcommercial sources. Typically, such vectors are provided containingconvenient restriction sites for insertion of the desired DNA segment.Examples of such vectors are pSVL and pKSV-10 (Pharmacia), pBPV-1, pML2d(International Biotechnologies), pTDT1 (ATCC® 31255) and othereukaryotic expression vectors.

Eukaryotic cell expression vectors used to construct the rDNA moleculesof the present invention may further include a selectable marker that iseffective in an eukaryotic cell, preferably a drug resistance selectionmarker. A preferred drug resistance marker is the gene whose expressionresults in neomycin resistance, i.e., the neomycin phosphotransferase(neo) gene. (Southern et al., J. Mol. Anal. Genet. 1:327-341 (1982)).Alternatively, the selectable marker can be present on a separateplasmid, the two vectors introduced by co-transfection of the host cell,and transfectants selected by culturing in the appropriate drug for theselectable marker.

To express the antibodies, or antibody portions of the invention, DNAsencoding partial or full-length light and heavy chains are inserted intoexpression vectors such that the genes are operatively linked totranscriptional and translational control sequences. Expression vectorsinclude plasmids, retroviruses, cosmids, YACs, EBV-derived episomes, andthe like. The antibody gene is ligated into a vector such thattranscriptional and translational control sequences within the vectorserve their intended function of regulating the transcription andtranslation of the antibody gene. The expression vector and expressioncontrol sequences are chosen to be compatible with the expression hostcell used. The antibody light chain gene and the antibody heavy chaingene can be inserted into separate vectors. In some embodiments, bothgenes are inserted into the same expression vector. The antibody genesare inserted into the expression vector by standard methods (e.g.,ligation of complementary restriction sites on the antibody genefragment and vector, or blunt end ligation if no restriction sites arepresent).

A convenient vector is one that encodes a functionally complete humanC_(H) or C_(L) immunoglobulin sequence, with appropriate restrictionsites engineered so that any V_(H) or V_(L) sequence can be easilyinserted and expressed, as described above. In such vectors, splicingusually occurs between the splice donor site in the inserted J regionand the splice acceptor site preceding the human C region, and also atthe splice regions that occur within the human C_(H) exons.Polyadenylation and transcription termination occur at nativechromosomal sites downstream of the coding regions. The recombinantexpression vector can also encode a signal peptide that facilitatessecretion of the antibody chain from a host cell. The antibody chaingene may be cloned into the vector such that the signal peptide islinked in-frame to the amino terminus of the antibody chain gene. Thesignal peptide can be an immunoglobulin signal peptide or a heterologoussignal peptide (i.e., a signal peptide from a non-immunoglobulinprotein).

In addition to the immunogenic polypeptides, Nogo receptor-1 antibodies,antigen-binding fragments, soluble Nogo receptor-1 polypeptides andsoluble Nogo receptor-1 fusion proteins of the present invention, therecombinant expression vectors of the invention carry regulatorysequences that control their expression in a host cell. It will beappreciated by those skilled in the art that the design of theexpression vector, including the selection of regulatory sequences maydepend on such factors as the choice of the host cell to be transformed,the level of expression of protein desired, etc. Preferred regulatorysequences for mammalian host cell expression include viral elements thatdirect high levels of protein expression in mammalian cells, such aspromoters and/or enhancers derived from retroviral LTRs, cytomegalovirus(CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (suchas the SV40 promoter/enhancer), adenovirus, (e.g., the adenovirus majorlate promoter (AdMLP)), polyoma and strong mammalian promoters such asnative immunoglobulin and actin promoters. For further description ofviral regulatory elements, and sequences thereof, see, e.g., U.S. Pat.No. 5,168,062 by Stinski, U.S. Pat. No. 4,510,245 by Bell et al. andU.S. Pat. No. 4,968,615 by Schaffner et al.

In one embodiment, a proprietary expression vector of Biogen IDEC, Inc.,referred to as NEOSPLA (U.S. Pat. No. 6,159,730) may be used. Thisvector contains the cytomegalovirus promoter/enhancer, the mouse betaglobin major promoter, the SV40 origin of replication, the bovine growthhormone polyadenylation sequence, neomycin phosphotransferase exon 1 andexon 2, the dihydrofolate reductase gene and leader sequence. Thisvector has been found to result in very high level expression upontransfection in CHO cells, followed by selection in G418 containingmedium and methotrexate amplification. Of course, any expression vectorwhich is capable of eliciting expression in eukaryotic cells may be usedin the present invention. Examples of suitable vectors include, but arenot limited to plasmids pcDNA3, pHCMV/Zeo, pCR3.1, pEF1/His, pIND/GS,pRc/HCMV2, pSV40/Zeo2, pTRACER-HCMV, pUB6/V5-His, pVAX1, and pZeoSV2(available from Invitrogen, San Diego, Calif.), and plasmid pCI(available from Promega, Madison, Wis.). Additional eukaryotic cellexpression vectors are known in the art and are commercially available.Typically, such vectors contain convenient restriction sites forinsertion of the desired DNA segment. Exemplary vectors include pSVL andpKSV-10 (Pharmacia), pBPV-1, pmI2d (International Biotechnologies),pTDT1 (ATCC 31255), retroviral expression vector pMIG and pLL3.7,adenovirus shuttle vector pDC315, and AAV vectors. Other exemplaryvector systems are disclosed e.g., in U.S. Pat. No. 6,413,777.

Other embodiments of the invention use a lentiviral vector forexpression of the polynucleotides of the invention, e.g., NgR antagonistpolynucleotides, e.g., siRNA molecules. Lentiviruses can infectnoncycling and postmitotic cells, and also provide the advantage of notbeing silenced during development allowing generation of transgenicanimals through infection of embryonic stem cells. Milhavet et al.,Pharmacological Rev. 55:629-648 (2003). Other polynucleotide expressingviral vectors can be constructed based on, but not limited to,adeno-associated virus, retrovirus, adenovirus, or alphavirus.

Transcription of the polynucleotides of the invention, e.g., siRNAmolecule sequences can be driven from a promoter for eukaryotic RNApolymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III(pol III). Transcripts from pol II or pol III promoters are expressed athigh levels in all cells; the levels of a given pol II promoter in agiven cell type depends on the nature of the gene regulatory sequences(enhancers, silencers, etc.) present nearby. Prokaryotic RNA polymerasepromoters are also used, providing that the prokaryotic RNA polymeraseenzyme is expressed in the appropriate cells (Elroy-Stein and Moss,Proc. Natl. Acad. Sci. USA 87:6743-7 (1990); Gao and Huang, NucleicAcids Res. 21:2867-72 (1993); Lieber et al., Methods Enzymol. 217:47-66(1993); Zhou et al., Mol. Cell. Biol. 10:4529-37 (1990)). Severalinvestigators have demonstrated that polynucleotides expressed from suchpromoters can function in mammalian cells (e.g. Kashani-Sabet et al.,Antisense Res. Dev. 2:3-15 (1992); Ojwang et al., Proc. Natl. Acad. Sci.USA 89:10802-6 (1992); Chen et al., Nucleic Acids Res. 20:4581-9 (1992);Yu et al., Proc. Natl. Acad. Sci. USA 90:6340-4 (1993); L'Huillier etal., EMBO J. 11:4411-8 (1992); Lisziewicz et al., Proc. Natl. Acad. Sci.U.S.A 90:8000-4 (1993); Thompson et al., Nucleic Acids Res. 23:2259(1995); Sullenger & Cech, Science 262:1566 (1993)). More specifically,transcription units such as the ones derived from genes encoding U6small nuclear (snRNA), transfer RNA (tRNA) and adenovirus VA RNA areuseful in generating high concentrations of desired RNA molecules suchas siRNA in cells (Thompson et al., supra; Couture and Stinchcomb, 1996,supra; Noonberg et al., Nucleic Acid Res. 22:2830 (1994); Noonberg etal., U.S. Pat. No. 5,624,803; Good et al., Gene Ther. 4:45 (1997);Beigelman et al., International PCT Publication No. WO 96/18736. ThesiRNA transcription units can be incorporated into a variety of vectorsfor introduction into mammalian cells, including but not restricted to,plasmid DNA vectors, viral DNA vectors (such as adenovirus oradeno-associated virus vectors), or viral RNA vectors (such asretroviral or alphavirus vectors) (for a review see Couture andStinchcomb, 1996, supra).

In addition to the heterologous genes and regulatory sequences, therecombinant expression vectors of the invention may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example,typically the selectable marker gene confers resistance to drugs, suchas G418, hygromycin or methotrexate, on a host cell into which thevector has been introduced. Preferred selectable marker genes includethe dihydrofolate reductase (DHFR) gene (for use in dhfr host cells withmethotrexate selection/amplification) and the neo gene (for G418selection).

Host Cells and Methods of Recombinantly Producing Protein of theInvention

Nucleic acid molecules encoding anti-Nogo receptor-1 antibodies,immunogenic peptides, soluble Nogo receptor-1 polypeptides, soluble Nogoreceptor-1 fusion proteins of this invention and vectors comprisingthese nucleic acid molecules can be used for transformation of asuitable host cell. Transformation can be by any known method forintroducing polynucleotides into a host cell. Methods for introductionof heterologous polynucleotides into mammalian cells are well known inthe art and include dextran-mediated transfection, calcium phosphateprecipitation, polybrene-mediated transfection, protoplast fusion,electroporation, encapsulation of the polynucleotide(s) in liposomes,and direct microinjection of the DNA into nuclei. In addition, nucleicacid molecules may be introduced into mammalian cells by viral vectors.

Transformation of appropriate cell hosts with a rDNA molecule of thepresent invention is accomplished by well known methods that typicallydepend on the type of vector used and host system employed. With regardto transformation of prokaryotic host cells, electroporation and salttreatment methods can be employed (see, for example, Sambrook et al.,Molecular Cloning—A Laboratory Manual, Cold Spring Harbor LaboratoryPress (1989); Cohen et al., Proc. Natl. Acad. Sci. USA 69:2110-2114(1972)). With regard to transformation of vertebrate cells with vectorscontaining rDNA, electroporation, cationic lipid or salt treatmentmethods can be employed (see, for example, Graham et al., Virolog52:456-467 (1973); Wigler et al., Proc. Natl. Acad. Sci. USA76:1373-1376 (1979)).

Successfully transformed cells, i.e., cells that contain a rDNA moleculeof the present invention, can be identified by well known techniquesincluding the selection for a selectable marker. For example, cellsresulting from the introduction of an rDNA of the present invention canbe cloned to produce single colonies. Cells from those colonies can beharvested, lysed and their DNA content examined for the presence of therDNA using a method such as that described by Southern, J. Mol. Biol.98:503-517 (1975) or the proteins produced from the cell may be assayedby an immunological method.

Host cells for expression of a polypeptide or antibody of the inventionfor use in a method of the invention may be prokaryotic or eukaryotic.Mammalian cell lines available as hosts for expression are well known inthe art and include many immortalized cell lines available from theAmerican Type Culture Collection (ATCC®). These include, inter alia,Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, babyhamster kidney (BHK) cells, monkey kidney cells (COS), humanhepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and a numberof other cell lines. Cell lines of particular preference are selectedthrough determining which cell lines have high expression levels. Otheruseful eukaryotic host cells include plant cells. Other cell lines thatmay be used are insect cell lines, such as Sf9 cells. Exemplaryprokaryotic host cells are E. coli and Streptomyces.

When recombinant expression vectors encoding the immunogenicpolypeptides, Nogo receptor-1 antibodies or antigen-binding fragments,soluble Nogo receptor-1 polypeptides and soluble Nogo receptor-1 fusionproteins of the invention are introduced into mammalian host cells, theyare produced by culturing the host cells for a period of time sufficientto allow for expression of the antibody, polypeptide and fusionpolypeptide in the host cells or, more preferably, secretion of theimmunogenic polypeptides, Nogo receptor-1 antibodies or antigen-bindingfragments, soluble Nogo receptor-1 polypeptides and soluble Nogoreceptor-1 fusion proteins of the invention into the culture medium inwhich the host cells are grown. Immunogenic polypeptides, Nogoreceptor-1 antibodies or antigen-binding fragments, soluble Nogoreceptor-1 polypeptides and soluble Nogo receptor-1 fusion proteins ofthe invention can be recovered from the culture medium using standardprotein purification methods.

Further, expression of immunogenic polypeptides, Nogo receptor-1antibodies or antigen-binding fragments, soluble Nogo receptor-1polypeptides and soluble Nogo receptor-1 fusion proteins of theinvention of the invention (or other moieties therefrom) from productioncell lines can be enhanced using a number of known techniques. Forexample, the glutamine synthetase gene expression system (the GS system)is a common approach for enhancing expression under certain conditions.The GS system is discussed in whole or part in connection with EuropeanPatent Nos. 0 216 846, 0 256 055, and 0 323 997 and European PatentApplication No. 89303964.4.

Host Cells

The present invention further provides host cells transformed with anucleic acid molecule that encodes a Nogo receptor-1 antibody,antigen-binding fragment, soluble Nogo receptor-1 polypeptide and/orsoluble Nogo receptor-1 fusion protein of the invention. The host cellcan be either prokaryotic or eukaryotic. Eukaryotic cells useful forexpression of a protein of the invention are not limited, so long as thecell line is compatible with cell culture methods and compatible withthe propagation of the expression vector and expression of the geneproduct. Preferred eukaryotic host cells include, but are not limitedto, yeast, insect and mammalian cells, preferably vertebrate cells suchas those from a mouse, rat, monkey or human cell line. Examples ofuseful eukaryotic host cells include Chinese hamster ovary (CHO) cellsavailable from the ATCC® as CCL61, NIH Swiss mouse embryo cells NIH-3T3available from the ATCC® as CRL1658, baby hamster kidney cells (BHK),and the like eukaryotic tissue culture cell lines.

Other useful eukaryotic host cells include plant cells. Other cell linesthat may be used are insect cell lines, such as Sf9 cells. Exemplaryprokaryotic host cells are E. coli and Streptomyces.

Production of Recombinant Proteins Using a rDNA Molecule

The present invention further provides methods for producing an a Nogoreceptor-1 antibody or antigen-binding fragment, soluble Nogo receptor-1polypeptide and/or soluble Nogo receptor-1 fusion protein of theinvention using nucleic acid molecules herein described. In generalterms, the production of a recombinant form of a protein typicallyinvolves the following steps:

First, a nucleic acid molecule is obtained that encodes a protein of theinvention. If the encoding sequence is uninterrupted by introns, it isdirectly suitable for expression in any host.

The nucleic acid molecule is then optionally placed in operable linkagewith suitable control sequences, as described above, to form anexpression unit containing the protein open reading frame. Theexpression unit is used to transform a suitable host and the transformedhost is cultured under conditions that allow the production of therecombinant protein. Optionally the recombinant protein is isolated fromthe medium or from the cells; recovery and purification of the proteinmay not be necessary in some instances where some impurities may betolerated.

Each of the foregoing steps can be done in a variety of ways. Forexample, the desired coding sequences may be obtained from genomicfragments and used directly in appropriate hosts. The construction ofexpression vectors that are operable in a variety of hosts isaccomplished using appropriate replicons and control sequences, as setforth above. The control sequences, expression vectors, andtransformation methods are dependent on the type of host cell used toexpress the gene and were discussed in detail earlier. Suitablerestriction sites can, if not normally available, be added to the endsof the coding sequence so as to provide an excisable gene to insert intothese vectors. A skilled artisan can readily adapt any host/expressionsystem known in the art for use with the nucleic acid molecules of theinvention to produce recombinant protein.

It will be readily apparent to one of ordinary skill in the relevantarts that other suitable modifications and adaptations to the methodsand applications described herein are obvious and may be made withoutdeparting from the scope of the invention or any embodiment thereof. Inorder that this invention may be better understood, the followingexamples are set forth. These examples are for purposes of illustrationonly and are not to be construed as limiting the scope of the inventionin any manner.

EXAMPLE 1 Production of Murine Monoclonal Anti-Nogo Receptor-1Antibodies

Anti-Nogo receptor-1 antibodies that specifically bind an immunogenicNogo receptor-1 polypeptide of the invention were made using thefollowing methods and procedures.

Immunizations

Two immunization approaches were used:

1. COS-7 Cells or Cell Membranes Containing Nogo Receptor-1 (NogoR-1) asthe Immunogen

The rat Nogo receptor-1 gene (GenBank™ No. AF 462390) was subcloned intothe mammalian expression vector pEAG1256 (Biogen®) that contained theCMV promotor and geneticin resistance gene for drug selection. Therecombinant plasmid was transfected into COS-7 cells using Superfect(Qiagen®). Transfectants were selected using geneticin (Gibco™, 2mg/ml), cloned and verified for surface expression of Nogo receptor-1protein by FACS. COS-7 membranes were prepared from these cellsaccording to procedures as described [Wang et al., J. Neurochem.75:1155-1161 (2000)] with two washings, and stored at 1 mg/ml [proteinconcentration] in 10% glycerol at −70° C.

Eight-week-old female RBF mice (Jackson Labs, Bar Harbor, Me.) wereimmunized intraperitoneally either with an emulsion containing 50 μg ratNogo receptor-1-COS-7 membranes or whole COS-7 cells expressing Nogoreceptor-1 on the surface and 50 μl RIBI MPL+TDM+CWS adjuvant (Sigma®Chemical Co., St. Louis, Mo.) once every two weeks (Lipman et al.,1992). Sera from the immunized mice were collected before the firstimmunization, 7 days after the second and third immunizations, and 38days after the third immunization and the anti-Nogo receptor-1 antibodytiters were measured by ELISA as described below.

2. Specific Nogo Receptor-1 Peptides as the Immunogen

The rat Nogo receptor-1 gene sequence was subjected to antigenicityanalyses using Vector NTi™ software (FIG. 2). Antigenic peptidesidentified in the analyses were conjugated to Keyhole Limpet Hemocyanin(KLH) using standard glutaraldehyde procedures.

Eight-week-old female RBF mice (Jackson Labs, Bar Harbor, Me.) wereimmunized intraperitoneally with an emulsion containing 50 μgKLH-conjugated peptides and 50 μl complete Freund's adjuvant (Sigma®Chemical Co., St. Louis, Mo.) once every two weeks. Serum from theimmunized mice was collected before the first immunization and 1 weekafter the second and third immunizations and anti-Nogo receptor-1antibody titers were measured. A booster dose was given after the thirdimmunization. Three days after this booster dose immunization, fusionexperiments were initiated.

Hybridoma Production and Screening

Sera from mice immunized with antigenic Nogo receptor-1 peptides werescreened by ELISA whereas sera from mice immunized with COS-7 cellsexpressing Nogo receptor-1 were screened by flow cytometry. Mice thatwere positive for antibodies that specifically bound Nogoreceptor-1-COS-7 cells were identified by flow cytometry and weresacrificed. Splenocytes were isolated from the mice and fused to theFlab 653 myeloma (an APRT-derivative of a Ig-/HGPRT-Balb/c mousemyeloma, maintained in DMEM containing 10% FBS, 4500 mg/L glucose, 4 mML-glutamine, and 20 mg/ml 8-azaguanine) as described (Kennett et al.,Monoclonal Antibodies: A New Dimension in Biological Analysis, PlenumPress, New York (1993)). Fused cells were plated into 24- or 48-wellplates (Corning Glass Works, Corning, N.Y.), and fed with adenine,aminopterin and thymidine containing culture media. AAT resistantcultures were screened by ELISA or flow cytometry for binding to eitherNogo receptor-1-COS-7 cells or to a Nogo receptor-1 antigenic peptide asdescribed below. Cells in the positive wells were further subcloned bylimiting dilution.

To screen for antibody binding to a Nogo receptor-1 antigenic peptide,the peptides that were used as immunogens were conjugated to BSA. 0.5 μgof the conjugated peptide in 50 μl of 0.1 M sodium bicarbonate buffer,pH 9.0 was added to each well of a 96-well MaxiSorp™ plate (Nunc™). Theplate was then incubated at 37° C. for 1 hour or 4° C. for 16 hours andnon-specific binding sites were blocked using 25 mM HEPES, pH 7.4containing 0.1% BSA, 0.1% ovalbumin, 0.1% blotto and 0.001% azide.Hybridoma supernatant was added and incubated at 25° C. for 1 hour.After washing three times with PBS, 501 of a 1:10,000 dilution ofhorseradish peroxidase-conjugated goat anti-mouse secondary antibody(Jackson ImmunoResearch Inc.) was added to each well and incubatedfurther for 1 hour. After three washings, color was developed by TMB(Pierce) and stopped with 2 M sulphuric acid. Color intensity wasmonitored in a spectrophotometer at 450 nm.

Antibodies were screened for binding to full length Nogo receptor-1 asfollows. COS-7 cells were labeled with 0.1 uM CellTracker™ Green CMFDA(Molecular Probes, Eugene, Oreg.) as described by the vendor. Equalvolumes of CellTracker™ labeled control cells were mixed with washedNogo receptor-1-COS-7 cells before incubation with anti-Nogo receptor-1test sera. Fifty microliters of the cell mixture was dispensed into eachwell of a 96-well V-bottom polystyrene plates (Costar® 3877, Corning,N.Y.) and 100 μl of hybridoma supernatant or a control anti-Nogoreceptor-1 antibody was added. After incubation at 4° C. for 30 minutes,the cells were washed and incubated with 50 μl ofR-phycoerythrin-conjugated affinity pure F(ab′)2 fragment goatanti-mouse IgG Fc gamma specific second:antibody (1:200, JacksonImmunoResearch Laboratory, West Grove, Pa.) in PBS. At the end of theincubation, the cells were washed twice with PBS and suspended in 200 μlof PBS containing 1% FBS, and subjected to FACS analyses. Alternately,Nogo receptor-1-COS-7 cells were mixed with hybridoma supernatant andthen treated with R-phycoerythrin-conjugated goat anti-mouse secondaryantibody and directly subjected to standard FACS analyses.

We generated 25 anti-Nogo receptor-1 antibodies using a variety ofimmunogens. We generated two antibodies, 7E11 and 5B10, using a peptidesequence corresponding to rat Nogo receptor-1 residues 110-125 as theimmunogen. We generated three antibodies, 1H2, 3G5 and 2F7, usingmembranes prepared from COS7 cells transfected with full length rat Nogoreceptor-1 as the immunogen. We generated 13 antibodies usingsNogoR310-Fc as the immunogen (1D9.3, 1E4.7, 1B4.3, 2C4.3, 1F10.3,2H1.4, 1H3.3, 1G4.1, 1E4.1, 2G7.1, 2C4.1, 2F11.1, and 1H4.1) and 7antibodies using a peptide sequence corresponding to rat Nogo receptor-1residues 423-434 as the immunogen (2E8.1, 2G11.2, and 1B5.1).

Sequence Analysis of Monoclonal Antibodies 7E11 and 5B10

We extracted total RNA using Qiagen® RNeasy® mini kit, and generatedcDNA from the isolated RNA. We amplified the light chain sequence by PCRusing primers 5′-TGAGGAGACGGTGACCGTGGTCCCTTGGCCCCAG-3′ (SEQ ID NO: 12)and 5′-AGGTSMARCTGCAGSAGTCWGG-3′ (SEQ ID NO: 25). We amplified the heavychain sequence by PCR using primers5′-GGGGATATCCACCATGAAGTTGCCTGTTAGGCTGTTG-3′ (SEQ ID NO: 13) and5′-GGGGATATCCACCATGAGGKCCCCWGCTCAGYTYCTKGGA-3′ (SEQ ID NO: 14). Theseprimers comprise degenerate nucleotides as follows: S represents G or C;M represents A or C, R represents G or A; W represents A or T; Krepresents G or T; and Y represents T or C. We cloned the PCR fragmentsinto a sequencing vector and determined the DNA sequence of the CDRs bydideoxychain termination using primers specific for the sequencingvector. We conceptually translated the DNA sequences and partial aminoacid sequences of the CDR regions of the heavy of light chains of themonoclonal antibodies 7E11 and 5B10 are shown in Table 2. The 3 CDRsfrom the heavy and light chains of the mAbs are underlined in Table 2.The light chains of 7E11 and 5B10 have 94% amino acid sequence identityand the heavy chains have 91% amino acid sequence identity. mAbs 7E11,5B10, and 1H2 are of the IgG1 isotype and mAbs 3G5 and 2F7 are of theIgG2a isotype. Each of these five mAbs has a light chain of the kappaisotype. We analyze the sequence of the other monoclonal antibodies bythis approach.

TABLE 2 AMINO ACID SEQUENCE OF MABS 7E11 AND 5B10 SEQ ID Sequence NO:7E11 MKLPVRLLVLMFWIPASSSDVVMTQTPLSLPVSLGDQASIS 15 LightCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGV ChainPDRFSGSGSGTDFTLKISRVDAEDLGVYFCSQSTHVPFTFG GGTKLEIKRADAAPTVSISHH 5B10MKLPVRLLVLMFWIPASSSDVVMTQTPLSLPVSLGDQASIS 16 LightCRSSQSLVHSNGYTYLHWYLQRPGQSPKLLIYKVSNRFSGV ChainPDRFSGSGSGTDFTLKISRVDAEDLGVYFCSQSTHVPFTFG GGTKLEIKRADAAPTVSISHH 7E11VQLQESGAELVMPGASVKMSCKASGYTFTDYWMHWVKQRPG 17 HeavyQGLEWIGAIDPSDSYSSYNQNFKGKATLTVDGSSSTAYMQL ChainSSLTSEDSAVYYCARRITEAGAWFAYWGQGTTVT 5B10LQXSGAEIVMPGTAVTMSCKASGYTFTDFWMHWVKQRPGQG 18 HeavyLEWIGAIDPSDSYSRINQKFKGKATLTVDESSSTAYMQLSS ChainLTSEDSAVYYCARRITEAGAWFAYWGQGTTVT

Epitope Mapping of Monoclonal Antibody 7E11

mAb 7E11 binds both rat and human NgR1. To determine the epitoperesponsible for 7E11 binding, we generated fragments and syntheticpeptides of rat NgR1 and tested them for 7E11 binding.

A recombinant fragment of the rat NgR1 that contains all 8 LRR domainsand the N- and C-terminal caps (sNgR310) was treated with either acid orcyanogen bromide (CNBr) and separated the fragments by gelelectrophoresis. Untreated sNgR310 migrates with an apparent molecularweight of 42 kDa. Acid treatment of sNgR310 produced two major cleavageproducts of 15 kDa (aa 27-aa 122) and 30 kDa (aa 123-aa 310). CNBrtreatment generated three fragments, a 33/35 kDa doublet (aa 27-aa 229),which may represent fragments with heterogeneous glycosylation, a 10 kDaproduct (aa 241-aa 310), and an 11-amino-acid fragment (aa 230-aa 240),which is not retained on the gel. A western blot of the gel was probedwith 7E11 and demonstrated that it bound to intact rat NgR1 (aa 27-aa310), the 15 kDa acid fragment (aa 27-aa 122) and the 35 kDa CNBrfragment (aa 27-aa 229). 7E11 did not bind to the 30 kDa acid fragment(aa 123-aa 310) or the 10 kDa CNBr fragment (aa 241-aa 310). Both the 15kDa acid fragment and the 35 kDa CNBr fragment contained the sequenceLDLSDNAQLRVVDPTT (SEQ ID NO: 1), consistent with 7E11 binding to asingle epitope on NgR1.

The 7E11 binding site was further analyzed by testing tryptic peptidedigests of sNgR310. HPLC analyses showed several fragments, indicatingthat there were several trypsin-sensitive lysine and arginine residuesin the NgR1 sequence. 7E11 bound only a single tryptic digest peptide,providing additional evidence that 7E11 binds to a single epitope onNgR1. Subsequent mass spectroscopy (MS) and sequence analyses identifiedthe bound peptide to be AAAFTGLTLLEQLDLSDNAQLR (SEQ ID NO: 26).

The LDLSDNAQLRVVDPTT peptide (SEQ ID NO: 1) was subjected to furthermapping analysis. The peptide was digested with trypsin, which yieldedtwo major fragments, LDLSDNAQLR (SEQ ID NO: 27) and VVDPTT (SEQ ID NO:28), and the ability of 7E11 to bind them was tested. MS analysisrevealed that the antibody bound peptide LDLSDNAQLR (SEQ ID NO: 27), andtherefore this peptide contains the binding epitope for 7E11. Withinthis peptide fraction, detailed MS analysis identified several scrambledpeptides that also bound 7E11, including peptides with deamination atAsn115 and Gln117, addition of Alanine at 112 or 113, or addition ofSerine at 114 (Table 3). These data indicate that several amino acidresidues located in this peptide fragment may not be critical for 7E11binding.

TABLE 3 MUTANT PEPTIDES BOUND BY 7E11. Peptides bound Amino AcidSequence Wild-type Fragment LDLSDNAQLR (SEQ ID NO: 27) DeaminatedLDLSDDAELR (SEQ ID NO: 29) Scrambled Fragment #1 LDLASDNAQLR (SEQ ID NO:30) Deaminated LDLASDDAELR (SEQ ID NO: 31) Scrambled Fragment #2LDALSDNAQLR (SEQ ID NO: 32) Deaminated LDALSDDAELR (SEQ ID NO: 33)Scrambled Fragment #3 LDLSSDNAQLR (SEQ ID NO: 34) Deaminated LDLSSDEARLR(SEQ ID NO: 35)

The LDLSDNAQLRVVDPTT (SEQ ID NO:1) peptide was also digested with theendoprotease Asp-N and 7E11 binding was tested. Endoprotease Asp-Ncleaved the peptide into 3 peptide fragments, L, DLS and DNAQLRVVDPTT(SEQ ID NO: 36). Of these products, 7 μl bound the DNAQLRVVDPTT (SEQ IDNO:36) peptide. Taken together, the trypsin and Asp-N cleavage datafurther localize the 7E11 binding epitope to the sequence shared betweenthem, DNAQLR (SEQ ID NO: 37).

The amino acid sequences of NgR1, NgR2, and NgR3 from various specieswere analyzed to predict critical residues in the 7E11 binding epitopebased on the observation that 7E11 bound rat and human NgR1 but notmouse NgR1, human NgR2 or mouse NgR3. Sequence alignment revealed thatamino acids 110-125 of rat NgR1 and the corresponding sequence of humanNgR1 are identical and that the mouse NgR1 sequence differs only by oneamino acid at position 119 (Arg119 in rat and human NgR1, and His119 inmouse NgR1; Table 4).

TABLE 4 SEQUENCE ALIGNMENT OF NGRS FROM DIFFERENT SPECIES. SEQ Sequenceof aa ID Protein(s) 110 to aa 119 NO: Rat & Human NgR1 LDLSDNAQLR 27Mouse NgR1 LDLSDNAQLH 38 Rat & Human NgR2 LDLGDNRHLR 39 Rat, Human& Mouse NgR3 LDLGDNRQLR 40

Arg119 on NgR1 contributes to 7E11 binding because it binds well to ratand human NgR1 but poorly to mouse NgR1. Similarly, because 7E11 doesnot bind well to NgR3, Ala116 is involved in the epitope because withinthe DNAQLR sequence (SEQ ID NO:37) NgR3 only differs from NGR1 by anArginine at the corresponding sequence. Within the DNAQLR (SEQ ID NO:37)sequence, 4 out of 6 of the residues in NgR2 are identical to rat NgR1.Ala 116 and Gln117 are replaced with Arginine and Histidine,respectively. This confirms that Ala 116 is an important amino acidresidue contributing to 7E11 binding, but does not necessarily precludethe involvement of Gln 117.

To verify these contact points, several peptides containing pointmutations within the LDLSDNAQLR sequence (SEQ ID NO: 27) were generatedand tested for 7E11 binding. The peptides were immobilized on aMaxiSorp™ plate (Nunc™ and serial dilutions of 7E11 were applied. Theresulting EC₅₀ values are shown in Table 5. 7E11 bound to mutantsLeu110Ala and Asp111Ala with similar EC₅₀ values as to the originalpeptide. When Gln17Ala was tested, the EC₅₀ increased 30-fold and whenArg119His was tested the EC₅₀ increased 25-fold. The most significantchange in EC₅₀ was observed when Arg119 was mutated to Alanine.

TABLE 5 7E11 BINDS TO MUTANT PEPTIDES WITH DIFFERENT EC₅₀ Change SEQ inID peptide Sequence EC₅₀ NO: No changes LDLSDNAQLRVVDPTT 0.55 1 L110AADLSDNAQLRVVDPTT 0.62 41 D111A LALSDNAQLRVVDPTT 0.31 42 Q117ALDLSDNAALRVVDPTT 16 43 R11911 LDLSDNAQLHVVDPTT 12 44 R119ALDLSDNAQLAVVDPTT 88 45

The position of the 7E11 binding epitope was also determined in therecently resolved crystal structure of sNgR310. As expected, thestructure shows that the 7E1 epitope is exposed on the surface of themolecule. Residues Arg119, Gln117, Ala116, and Asp114 protrude outwardfrom the structure while Leu118 and Asn 115 are located inward. Theepitope falls on top of an acidic patch within the concave surface ofthe structure and a basic surface that faces one of the sides.

Inhibition of Ligand Binding to Soluble Nogo Receptor-1 by MonoclonalAnti-Nogo Receptor-1 Antibody

The anti-Nogo receptor-1 monoclonal antibodies produced as describedabove were tested to determine whether they inhibited ligand binding toNogo receptor-1.

0.5 μg of a soluble Nogo receptor-1 fusion protein comprising amino acidresidues 26-344 of rat Nogo receptor-1 and the hinge and Fc region ofthe rat IgG1 molecule (sNogoR344-Fc) produced as described below wasimmobilized on 250 μg of protein-A- or wheatgerm agglutinin-conjugatedSPA beads (Amersham Pharmacia Biotech) for 2 hours at 25° C. SPA beadscoupled with Fc-sNogoR-1, anti-Nogo receptor-1 mAb and 1 μl ¹²⁵I-Nogo66(Amersham, 2000 Ci/mmol, 1 nM) in 50 μl of the HEPES-buffered incubationmedium (10 mM HEPES, pH 7.4, 0.1% bovine serum albumin, 0.1% ovalbumin,2 mM MgCl₂, 2 mM CaCl₂ and protease inhibitors) was added to each samplewell. After 16 hours, radioactivity was measured in quadruplicatesamples using a TopCount® (Packard). IC₅₀ values were calculated from acurve-fit analysis (FIG. 3) (PRISM, GraphPad Software, N.J.). In someexperiments, we also used AP-ligand conjugates (e.g. AP-Nogo66) anddetected binding by monitoring alkaline phosphatase activity. We alsoassayed the ability of the mAbs to block binding of the ligands MAG-Fcand AP-OM-gp to Nogo receptor-1.

Monoclonal antibodies 7E11, 5B10, 1H2, 3G5 and 2F7 all inhibited bindingof Nogo66, MAG and OM-gp to sNogoR344-Fc. The calculated IC₅₀ for Nogo66for 7E11 and 1H2 were 400 nM and 60 nM, respectively. The data fromELISAs monitoring mAb-mediated inhibition of binding of the threeligands to Nogo receptor-1 are summarized in Table 6.

TABLE 6 MABS INHIBIT BINDING OF NOGO66, MAG AND OM-GP TO NOGORECEPTOR-1. MAG + Nogo66 + OM-gp + mAb sNogoR344-Fc sNogoR344-FcsNogoR344-Fc 7E11 30 nM (60%) EC₅₀ = 1.7 μM EC₅₀ = 150 nM EC₅₀ = 0.5 μM1H2 30 nM (60%) ND ND 3G5 30 nM (60%) EC₅₀ = 9 nM ND 2F7 30 nM (55%)EC₅₀ = 10 nM EC₅₀ = 5 nM 1D9.3 30 nM (70%) EC₅₀ = 13 nM EC₅₀ = 5.2 nMEC₅₀ = 2.7 nM 2G7.1 30 nM (84%) EC₅₀ = 18 nM EC₅₀ = 1 nM 1E4.1 30 nM(75%) — EC₅₀ = 9.1 nM EC₅₀ = 2.8 nM 1G4.1 30 nM (90%) — EC₅₀ = 8.2 nMEC₅₀ = 9.9 nM 2C4.1 30 nM (50%) — ND 2F11.1 30 nM (45%) ND ND 1H4.1 — NDND 2E8.1 30 nM (87%) EC₅₀ = 1.5 nM EC₅₀ = 42.9 nM EC₅₀ = 9.2 nM 2G11.230 nM (80%) ND ND 1B5.1 30 nM (0%) ND ND The percent displacement isshown at 30 nM antibody and the EC₅₀ for certain mAbs determined fromcurve-fit analysis as described. “—” indicates no detectable activityand “ND” indicates not determined.

EXAMPLE 2 Production of Fab-Phage Anti-Nogo Receptor-1 Antibodies

Anti-Nogo receptor-1 Fab-phage antibodies that specifically bind animmunogenic Nogo receptor-1 polypeptide of the invention were also madeby screening a Fab-phage library as follows.

The MorphoSys Fab-phage library HuCAL® GOLD was screened againstrecombinant rat soluble sNogoR310-Fc protein and COS7 cells expressingrat Nogo receptor-1. Fab-phages that specifically bound to Nogoreceptor-1 were purified and characterized. The heavy chain of 14D5 isderived from the V_(H)2 gene and the light chain is derived from theV_(K)1 gene. The amino acid sequences of the CDRs of the heavy chain andlight chain of one of these Fab-phages, 14D5, are shown in Table 7.

TABLE 7 AMINO ACID SEQUENCE OF CDRS OF 14D5 SEQ ID Amino Acid SequenceNO: Heavy Chain CDR1 GFSLSTSGGSVG 19 Heavy Chain CDR2 LIYSNDTKYYSTSLKT20 Heavy Chain CDR3 SRFWTGEYDV 21 Light Chain CDR1 RASQNIAITLN 22 LightChain CDR2 LASSLQS 23 Light Chain CDR3 QQYDNYPL 24

14D5 binds to rat Nogo receptor-1 in both monovalent and bivalent forms.In addition, 14D5 binds to mouse and human Nogo receptor-1 and humanNogo receptor-2 but not mouse Nogo receptor-3.

EXAMPLE 3 Immunoprecipitation of Nogo Receptor-1 by Anti-Nogo Receptor-1Monoclonal Antibodies

To perform the immunoprecipitation, 100 μl lysed cells or 50 μl PiPLCtreated cells were mixed with 400 or 450 μl extraction buffer [10 mMTris-HCl, pH 7.2, 0.5% Tween-20™, 0.2 mM PMSF] or RIPA buffer,respectively in the presence of 30 μl Protein A or G and 1-2 μgantibody. The mixture was incubated in a shaker at 4° C. for 16 hours.

Samples were spun gently to pellet the protein A or G coupled beads. Thebeads were washed three times with 1 ml wash buffer (10 mM Tris-HCl, pH7.2, 0.1% Tween-20™). The final wash was performed using 10% of originalwash buffer.

Beads were resuspended in 100 μl of 2×SDS with 10% beta-mercaptoethanol.Samples were incubated at room temperature before being run on a 4-20%Tris-Glycine gel for SDS-PAGE. As determined by SDS-PAGE gel analysis,monoclonal antibodies, 3G5 and 2F7, immunoprecipitate Nogo receptor-1.

EXAMPLE 4 Determining Antibody Specificity by ELISA

To determine the specificity of the monoclonal and Fab-phage antibodiesproduced in Examples 1 and 2, we performed an ELISA using a panel ofNogo receptor-1 polypeptides. The panel consisted of sNogoR310-Fc (afusion protein comprising amino acids 26-310 of rat Nogo receptor-1 anda rat Fc fragment), sNogoR344-Fc (see supra), polypeptide p-617 (SEQ IDNO: 1), polypeptide p-618 (a 19-amino acid polypeptide from the LRR7region of rat Nogo receptor-1; FIG. 2; SEQ ID NO: 11) and polypeptidesp-4 and p-5 (polypeptides from the LRR5 and LRRCT regions of Nogoreceptor-1, respectively). Ovalbumin and BSA were used as controls. Asshown in FIG. 4, in Abs 1H2, 3G5 and 2F7 all specifically bound tosNogoR344-Fc. In similar experiments, those antibodies also specificallybound a polypeptide consisting of amino acids 310-344 of rat Nogoreceptor-1 (SEQ ID NO: 3) and mAbs 7E11 and 5B10 specifically boundpolypeptide p-617 (SEQ ID NO: 1).

Ten of the antibodies (1D9.3, 1E4.7, 1B4.3, 2C4.3, 1F10.3, 2H1.4, 1R3.3,1G4.1, 1E4.1, and 2G7.1) from the sNogoR310-Fc immunization displacedeach other for binding, indicating that they recognize a similar oroverlapping epitopes on sNogoR310-Fc. The other three antibodies fromthe sNogoR310-Fc immunization (2C4.1, 2F11.1, and 1H4.1) recognizedifferent epitopes located in amino acid residues 26-310.

We also performed ELISA binding assays using the Fab-phage 14D5. WhereAP-Nogo66, AP-OM-gp and MAG-Fc ligands were allowed to bind toimmobilized sNogoR344-Fc, 1 μM 14D5 completely inhibited Nogo and MAGbinding. 10 μM of 14D5 was required to completely inhibit the binding ofOM-gp to sNogoR344-Fc.

EXAMPLE 5 Neurite Outgrowth Assay

To test the ability of the monoclonal and Fab-phage antibodies producedabove to lessen the inhibitory effect of CNS myelin on neurons, Lab-Tek®culture slides (4 wells) were coated with 0.1 mg/ml poly-D-lysine(Sigma®). CNS myelin or PBS was spotted as 3 μl drops. Fluorescentmicrospheres (Polysciences) were added to the myelin/PBS to allow lateridentification of the drops (Grandpre et al., Nature 403:439-444(2000)). Lab-Tek® slides were then rinsed and coated with 10 μg/mllaminin (Gibco™). Dorsal root ganglions (DRG's) from P3-4 Sprague Dawleyrat pups were dissociated with 1 mg/ml collagenase type 1 (Worthington),triturated with fire-polished Pasteur pipettes-pre-plated to enrich inneuronal cells and finally plated at 23,000 cells/well on the pre-coatedLab-Tek® culture slides. The culture medium was F12 containing 5% heatinactivated donor horse serum, 5% heat inactivated fetal bovine serumand 50 ng/ml mNGF and incubated at 37° C. and 5% CO₂ for 6 hours.Fifteen μg/ml of mAb 7E11 was added immediately after plating.

Slides were fixed for 20 minutes with 4% paraformaldehyde containing 20%sucrose and stained for the neuronal marker anti beta-III-tubulin(Covance TUJ1) diluted 1:500. As secondary antibody anti-mouse AlexaFluor® 594 (Molecular Probes) was diluted 1:300 and slides werecoverslipped with Gel/Mount™ (Biomeda™). 5× digital images were acquiredwith OpenLab™ software and analysed by using the MetaMorph® software forquantification of neurite outgrowth.

MAb 7E11 protected DRG neurons from myelin-mediated inhibition ofneurite outgrowth. (FIG. 5). Similar results were observed with mAbs 1H2and 3G5.

In a neurite outgrowth protection assay where rat P7 DRG neurons werecultured on a CNS myelin substrate, bivalent 14D5 also efficientlypromoted neurite outgrowth.

EXAMPLE 6 Immunohistochemistry with 7E11 on Cells Transfected with NogoReceptor-1

To further characterize the binding properties of anti-Nogo receptor-1mAbs produced as described in Example 1, we compared binding to bothfixed and live COS-7 or 293 cells expressing rat or human Nogoreceptor-1.

Fixed Cells:

Nogo receptor-1 transfected and non-transfected cells were plated in8-well Lab-Tek® culture slides, fixed with 4% paraformaldehyde for 15minutes, blocked with 10% normal goat serum, 0.1% Triton X-100 in PBSfor 1 hour. Mab 7E11 was added at 15 μg/ml and 1.5 μg/ml in blockingsolution and incubated for 2 hours at room temperature;Alexa®-conjugated secondary antibody anti-mouse (Molecular Probes) wasincubated at a 1:300 dilution in blocking solution for 1 hour; DAPI wasadded at 5 μg/ml to the secondary antibody to label all nuclei.

Live Cells:

Transfected and non-transfected cells were plated in 8 well Lab-Tek®culture slides, blocked with FACS buffer (containing 4% donor horseserum) for 30 minutes at 4° C., incubated with 7E11 at 15 μg/ml and 1.5μg/ml in FACS buffer for 1 hour at 4° C., rinsed and incubated withsecondary antibody anti-mouse-Alexa® (1:300 in FACS buffer) for 30minutes at 4° C.

Immunohistochemical staining experiments demonstrated that all of themAbs bound cells expressing rat Nogo receptor-1. mAbs 7E11, 2G7.1 and2C4.1 bound both fixed and live cells expressing human Nogo receptor-1.

EXAMPLE 7 Mouse Model of Spinal CORC Contusive Injury

To test the effect of anti-Nogo receptor-1 mAbs produced in Example 1 onneurons in vivo, we use a mouse spinal cord contusion injury model.

Female mice (18-22 g) are treated prophylactically with analgesic andantibiotic agents. Mice are anesthetized and placed in a stereotaxicapparatus with vertebral column fixation under a stereomicroscope.Trauma to the spinal cord is introduced by a modified version of theweight-drop method (M. Li et al., Neuroscience 99:333-342 (2000).

Briefly, a T9 and T10 laminectomy is made and the vertebral column isstabilized using a pair of mouse transverse clamps supporting the T9-T10transverse processes bilaterally. A stainless steel impact rod with adiameter of 1.4 mm and weight of 2 g, is raised 2.5 cm above the duraand dropped onto the spinal cord at the T10 level. During the surgery,mice are kept on a 37° C. warming blanket and 1 ml of warmed sterilesaline is administered subcutaneously to each mouse after surgery toavoid dehydration. The bladder is manually expressed once daily untilreflexive bladder control is regained.

All animals receive post-operative analgesia every 8-12 hours aftersurgery and antibiotic treatment twice daily for 7 days thereafter.Animals have free access to food and water for the duration of thestudy. Anti-Nogo receptor-1 antibodies are delivered to the injury sitevia intrathecal injection for 28 days as described in the rat spinalcord transection model below.

EXAMPLE 8 Characterization of Soluble Nogo Receptor-1 Fusion Proteins

To characterize soluble Nogo receptor-1 polypeptides (sNogoR-1) andfusion proteins (Fc-sNogoR-1) we performed the following experiment.

Three μg of soluble Nogo receptors (sNogoR310-Fc and sNogoR344-Fc) wereimmobilized on 250 μg WGA-SPA beads and received 0.5 μL of radioactiveligand (final concentration 0.5 nM) in a final volume of 100 μL ofbinding buffer (20 mM HEPES, pH 7.4, 2 mM Ca, 2 mM Mg, 0.1% BSA, 0.1%ovalbumin and protease inhibitors). Ligands included 10 μM Nogo66, 10 μM¹²⁵I-Nogo40 (amino acids 1-40 of NogoA) and 10 μL of anti-Nogoreceptor-1 antibody supernatant for each ligand set. The three tyrosineson Nogo40 were separately iodinated and designated as Nogo40-A, -B and-C respectively. Mean values of triplicates are presented as normalized% bound radioactivity (FIGS. 6, 7 and 8). Error bars indicate SEM. Boundradioactivity in the absence of inhibitors was taken as 100% and thelowest bound radioactivity in the presence of 10 μM Nogo40 was taken asthe 0% for data normalization.

EXAMPLE 9 Inhibition of Ligand Binding to Soluble Nogo Receptor-1 FusionProtein

A binding assay similar to the binding assay of Example 8 was used totest the ability of two mAbs produced in Example 1 to inhibit¹²⁵I-Nogo66 binding to sNogoR344-Fc. Mabs 2F7 and 3G5 inhibited¹²⁵I-Nogo66 binding to sNogoR344-Fc.

EXAMPLE 10 Neurite Outgrowth Assay

Lab-Tek® culture slides (4 wells) were coated with 0.1 mg/mlpoly-D-lysine (Sigma®). CNS myelin alone or mixed with sNogoR310,sNogoR310-Fc fusion protein, mAb 5B10 or control PBS were separatelyspotted as 3 μl drops. Fluorescent microspheres (Polysciences) wereadded to the myelin/PBS to allow later identification of the drops(Grandpre et al., Nature 403:439-444 (2000)). Lab-Tek® slides were thenrinsed and coated with 10 μl/ml laminin (Gibco™).

Dorsal root ganglions (DRG's) from P34 Sprague Dawley rat pups weredissociated with 1 mg/ml collagenase type 1 (Worthington), trituratedwith fire-polished Pasteur pipettes pre-plated to enrich in neuronalcells and finally plated at 23,000 cells/well on the pre-coated Labtekculture slides. The culture medium was F12 containing 5% heatinactivated donor horse serum, 5% heat inactivated fetal bovine serumand 50 ng/ml mNGF and incubated at 37° C. and 5% CO₂ for 6 hours.

Slides were fixed for 20 minutes with 4% paraformaldehyde containing 20%sucrose and stained for the neuronal marker anti beta-III-tubulin(Covance TUJ1) diluted 1:500. As secondary antibody anti-mouse AlexaFluor® 594 (Molecular Probes) was diluted 1:300 and slides werecoverslipped with Gel/Mount™ (Biømeda™). 5× digital images were acquiredwith OpenLab™ software and analyzed by using the MetaMorph® software forquantification of neurite outgrowth.

sNogoR310, sNogoR310-Fc and mAb 5B10 all protected DRG neurons frommyelin-mediated inhibition of neurite outgrowth (FIGS. 9-11). sNogoR310was used in a similar assay using chick neurons and was found to beprotective.

We also tested the neuro-protective effect of soluble Nogo receptors byperforming experiments with cells grown in the presence and absence oflaminin. Neuronal cell growth in media without laminin is poor andmodels neuronal stress conditions.

DRG's were dissected from post-natal day 6-7 rat pups (P6-7),dissociated into single cells and plated on 96-well plates pre-coatedwith poly-D-lysine as described above. In some wells 2 μg/ml laminin wasadded for 2-3 hours and rinsed before the cells were plated. After an18-20 h incubation the plates were fixed with 4% para-formaldehyde,stained with rabbit anti-Beta-III-tubulin antibody diluted 1:500(Covance®) and anti-HuC/D diluted 1:100 (Molecular Probes), andfluorescent secondary antibodies (Molecular Probes) were added at 1:200dilution. The ArrayScan® II (Cellomics®) was used to capture 5× digitalimages and to quantify neurite outgrowth as average neuriteoutgrowth/neuron per well, by using the Neurite outgrowth application.Nine 5× images from 3 wells/condition were analyzed.

In some experiments, a sub-clone of PC12 cells (Neuroscreen™) was used(Cellomics®). The Neuroscreen™ cells were pre-differentiated for 7 dayswith 200 ng/ml NGF, detached and replated on 96-well plates pre-coatedwith poly-D-lysine. In some wells 5 μg/ml laminin was added for 2-3hours and rinsed before the cells were plated. After 2 days incubationthe plates were fixed with 4% paraformaldehyde, stained with rabbitanti-Beta-III-tubulin antibody diluted 1:500 (Covance®) and Hoechst(nuclear stain). The ArrayScan® II was used to quantify neuriteoutgrowth as in the DRG cells.

sNogoR344-Fc or rat IgG were added in solution to P6-7 DRG neurons andto differentiated Neuroscreen™ cells at the time of plating.

The neuro-protective effect of sNogoR344-Fc was observed at 1 μM and 10μM when P6 DRG neurons were grown in the absence of laminin.Quantification of neurite outgrowth showed a dose-dependent increasewith the addition of sNogoR344-Fc. Addition of sNogoR344-Fc at the sameconcentrations to DRG neurons growing on a laminin substrate, did notproduce any unusual effect, indicating that sNogoR344-Fc is only activeon stressed cells. The neuro-protective effect of sNogoR344-Fc at thesame concentrations in the absence of laminin also was seen withNeuroscreen™ cells.

EXAMPLE 11 Production and Purification of Fc-sNogoR-1 Fusion Protein

A cDNA construct encoding amino acids 1-310 of rat Nogo receptor-1 wasfused to rat IgG1 Fc contained in a mammalian expression vector and thisvector was electroporated into Chinese hamster ovary (CHO) (DG44) cells.Cells were maintained in alpha-MEM, supplemented with 10% dialyzed fetalbovine serum, 2 mM glutamine and antibiotic-antimycotic reagents. Twodays after transfection, the conditioned media was collected andanalyzed by Western blot under reducing conditions. A protein band about60 kDa was detected using a polyclonal rabbit anti-Nogo receptor-1antibody. Cells were expanded and sorted using a R-PE conjugated goatanti-rat IgG antibody. After the second sorting, cells were plated at adensity of one cell/well in 96-well plates. Secreted soluble Nogoreceptor-1 protein levels from individual wells was tested and comparedusing a Sandwich ELISA. ELISA plate was coated with goat anti-rat IgGFcκ specific antibody. Conditioned media was applied. The bound solubleNogo receptor-1 protein was detected by HRP conjugated donkey anti-ratIgG Fab, Fc-specific antibody. Clone 4C12 had the highest secretionlevel. 4C12 was expanded and grown in CHO-M7 media in spinner flask. Thesecretion level was about 10 mg/L at 37° C.

CHO cells expressing the sNogoR310-Fc fusion protein were cultured inlarge scale. 1.7 L of concentrated conditioned media was obtained from a10 L bioreactor run. The pH was raised by addition of one-tenth volume1.0 M Tris-HCl, pH 8.9. Solid sodium chloride and glycine were added to3.0 M and 1.5 M respectively. A 60 mL protein A-Sepharose columnequilibrated with 10 mM Tris-HCl, 3 M sodium chloride, 1.5 M glycine, pH8.9 was prepared. Concentrated conditioned media was applied to thecolumn at 1.5 mL/min using a peristaltic pump. The column was washedwith 300 mL of 10 mM Tris-HCl, 3 M sodium chloride, 1.5 M glycine, pH8.9 followed with 120 mL 5 mM Tris-HCl, 3 M sodium chloride, pH 8.9.Protein was eluted with 25 mM sodium phosphate, 100 mM sodium chloride,pH 2.8. 10 mL fractions were collected in tubes containing 1.0 mL of 1.0M HEPES, pH 8.5. Protein fractions were pooled and dialyzed against 3×2L of 5 mM sodium phosphate, 300 mM NaCl, pH 7.4.

EXAMPLE 12 Spinal Cord Transection Assay

To test their ability to promote functional recovery in vivo, ansNogoR-1 fusion protein was tested in a rat spinal cord transectionassay.

Alzet® osmotic pumps were loaded with test solution (sNogoR310-Fc inPBS) made up freshly on the day of use. The loading concentration wascalculated to be 5 and 50 μM. Pumps were primed for >40 hours at 37° C.prior to implantation into animals. Female Long Evans rats were givenpre-operative analgesia and tranquilizer and anesthetized usingisoflurane (3% in O₂).

Rats were placed in a stereotaxic frame and the motor cortex exposed forinfusion of the tract tracing agent BDA (10,000 MW) bilaterally. Ratsthen undervent dorsal hemisection of the spinal cord at T5-T6 followedby implantation of the intrathecal catheter and pump system to delivertest compound (n=11 per group).

Rats were allowed to recover and survive up to 28 days after surgery.Behavioral scoring using the BBB system was recorded up to 28 days afterinduction of injury, just prior to termination of the in-life phase ofthe study. Following perfusion and fixation, spinal cords were removed,cryoprotected, sectioned, stained and axonal counts performed.

The Basso-Beattie-Bresnahan (BBB) locomotor rating scale (Basso et al.,Neurotrauma 13:343-359 (1996)), the inclined plane test and the inclinedgrid walking test (Li and Strittmatter, J. Neurosci. 23:4219-27 (2003))were monitored in rats and mice after injury. For the inclined planetest, we measured the maximal angle to which a 50 cm×60 cm board couldbe angled for 5 sec without the mouse sliding off. For inclined gridwalking, the mice were trained to climb a wire grid (35 cm long with2.54 cm squares) at a slope of 45 degrees. The number of instances inwhich the hindpaw dropped below the grid plane was scored for eachexcursion from bottom to top. For the rat behavioral testing, BBBlocomotor scale, grid walking and footprint analysis were performed. Forgrid walking, the rats were trained to walk on a wire grid (70 cm longwith 2.54 cm squares), and the number of instances in which the hindpawdropped below the grid plane was counted. For footprint analysis, thewalking patterns of rat hindpaws were recorded with ink during acontinuous locomotion across a 90 cm runway, and stride length on eachside and stride width were calculated (Metz et al., Brain Res.883:165-177 (2000)). All of these behavioral tests were performed by atleast two individuals. Throughout the surgery, behavioral testing andhistologic analysis, researchers were blind to the identity of thecompound in the minipump.

sNogoR310-Fc promoted functional recovery (FIG. 12).

EXAMPLE 13 Rat Spinal Cord Contusion Assay

The effect of soluble Nogo receptor-1 polypeptides and fusion proteinson neurons in vivo are tested in a rat spinal cord contusion assay.

Female hooded Long Evans rats (170-190 g) are treated prophylacticallywith analgesic and antibiotic agents. Ten minutes before surgery,animals are tranquilized with 2.5 mg/kg Midazolam i.p. and anesthetizedin 2-3% isoflurane in O₂. Rats are then shaved, wiped down with alcoholand betadine, and ocular lubricant applied to their eyes. Next, anincision is made down the midline and the T7 to T12 vertebrae exposed.

A dorsal laminectomy is performed at T9½ and T10 to expose the cord. Therat is mounted on the Impactor. T7 and T8 segments are first clamped andthen the T11 and T12 segments are attached to the caudal clamp. A softmaterial is placed underneath the chest of the rat. The Impactor rod isset to the zero position and the electrical ground clip is attached tothe wound edge. The Impactor rod is then raised to 25.0 mm andappropriately adjusted to a position directly above the exposed spinalcord. Next, the Impactor rod is released to hit the exposed cord and theImpactor rod is immediately lifted.

The rat is then dismounted, and Gelfoam® placed on the wound. The muscleover the wound is sutured, and the incision is surgically stapled.Animals are placed in an incubator until they recover from anesthesia.Rats are given antibiotics, analgesics, and saline as required. Bladdersare expressed every morning and evening thereafter until function isrecovered.

Soluble Nogo receptor-1 fusion protein (e.g., sNogoR310-Fc) isadministered intrathecally as described in the rat spinal cordtransection model above. BBB scoring is performed one-day after surgery,then every week thereafter until 4 to 6 weeks.

EXAMPLE 14 Expression of sNogoR310 in Transgenic Mice

We produced transgenic mice expressing soluble Nogo receptor-1 proteinto test its effect when expressed in vivo.

We cloned the mouse sNogoR310 cDNA (corresponding to amino acids 1-310of the Nogo receptor-1) into the NotI site of the C-3123 vector. In thisvector, sNogoR310 expression is under the control of the glialfibrillary acidic protein (gfap) gene regulatory elements, which allowhigh level expression with enhanced secretion from reactive astrocytesat site of injury. We digested the resulting vector sequentially withAatII and SfiI and isolated the gfap::sNogoR310 construct on a 3.4 kbfragment. We microinjected this fragment into embryos to generatetransgenic mice. We verified by PCR that the transgene had integratedand identified five founder lines. We crossed heterozygous males of thetwo founder lines with the highest expression levels to female C57BL/6Jmice. We confirmed that the GFAP-positive cells express and secretesNogoR310 in heterozygous transgenic mice by Western blot analysis usingantibody raised against Nogo receptor-1.

We homogenized the cortex and spinal cord in Tris-buffered salinesupplemented with protease inhibitors (Roche) and centrifuged thehomogenate at 40,000 rpm for 20 min at 4° C. We treated the supernatantwith 4% paraformaldehyde for 20 min to enhance antibody specificity anddialyzed prior to immunoblotting. We homogenized the particulatefraction by sonication in RIPA buffer (1% Triton® X-100, 0.5% sodiumdeoxycholate, 0.1% SDS in PBS), centrifuged the resulting homogenate andtreated this supernatant (detergent-soluble particulate fraction) asabove. We analyzed 20 μg of brain or spinal cord protein by immunoblotusing rabbit antiserum raised against Nogo receptor-1 at 1:2000dilution. We visualized immunoreactivity by incubation withAP-conjugated anti-rabbit IgG and NBT/BCIP AP substrates.

We detected secreted 37 kDa sNogoR310 in detergent-free soluble extractsof cortex and spinal cord from the two transgenic lines Tg08 and Tg01,but little if any soluble Nogo receptor-1 protein at 37 or 81 kDa ispresent in littermate wild type (WT) mice. Examination of theparticulate fractions demonstrated that there were comparable levels ofendogenous Nogo receptor-1 in both WT and transgenic mice.

EXAMPLE 15 Expression of sNogoR310 in Transgenic Mice After Injury

We tested the effect of CNS injury on sNogoR310 expression in transgenicmice by performing a dorsal over-hemisection injury. We obtainedsNogoR310 transgenic and nontransgenic control animals by matingheterozygous males with C57/BL6 females as described in Example 14.

We deeply anesthetized adult female heterozygous transgenic orlittermate WT mice (10-16 weeks of age) and performed a completelaminectomy, fully exposing the dorsal part of spinal cord at T6 and T7levels. We performed a dorsal over-hemisection at T6 with a 30-gaugeneedle and a pair of microscissors to completely sever the dorsal anddorsolateral corticospinal tracts (CSTs). We passed a marked needleacross the dorsal part of the spinal cord several times to assure thatthe lesion was at a depth of 1.0 mm. We sutured the muscle layers overthe laminectomies and closed the skin on the back with surgical staples.To trace the corticospinal tracts, we made a burr hole overlyingcerebral cortex on the right side into the skull 14 days after spinalcord injury. We applied the tracer BDA (MW 10,000, 10% in PBS)(Molecular Probes, Eugene, Oreg.) to 4 injection sites at a depth of 0.7mm from the cortical surface. Four weeks after injury, the mice wereperfused transcardially with PBS, followed by 4% paraformaldehyde. Miceused for sNogoR310 expression experiments did not receive any tracerinjection.

For the mice used for western blot analysis, the spinal cord at a levelbetween T3 and L3 was collected without perfusion 14 days after injury.Mice used for Nogo receptor-1 immunohistochemical staining were perfusedwith 4% paraformaldehyde 10 days after hemisection, and the injuredspinal cord was removed for sectioning. To examine sNogoR310 expressionin the injured brain of transgenic and WT mice, a cortex stab injury wasperformed with a number 11 scalpel blade held in a stereotaxic apparatus(David Kopf, Tujunga, Calif.). A 4 mm parasagittal cut was made, 0.5 mmposterior to Bregma, 1.5 nm laterally from midline and 3.5 mm deep.

We detected increased levels of sNogoR310 in soluble extracts of spinalcords ten days after the injury in transgenic mice but not in WT mice,consistent with the upregulation after injury of GFAP around the lesion.To confirm that this was not due to compensatory upregulation of Nogo-A,we tested its expression and found that it was similar in either intactor injured cortex and spinal cord from either WT and transgenic mice.

We examined the cellular expression of sNogoR310 in injured CNS byimmunostaining the injured brain and spinal cord containing the lesionarea with antibodies against Nogo receptor-1 and GFAP. The generalmorphology of reactive astrocytic glia does not differ between WT andtransgenic mice, but the density stained for Nogo receptor-1 in bothintra- and extracellular space is remarkably higher in thegfap::sNogoR310 transgenic mice than in WT mice, indicating increasedsNogoR310 expression around the lesion in transgenic mice. Nogoreceptor-1 protein is co-localized with astrocytic marker GFAP only inthe transgenic mice. There is also a greatly enhanced diffusenon-cellular staining in the transgenic samples, consistent withsNogoR310 in the extracellular space. Neuronal cell body Nogo receptor-1staining is detected in both WT and transgenic mice.

EXAMPLE 16 Secreted sNogoR310 Induces CST Sprouting in Transgenic Mice

We tested whether increased expression of sNogoR310 around the lesion intransgenic mice results in the regeneration of injured axons.

We investigated the integrity of descending corticospinal tracts (CST)by injecting anterograde tracer biotin dextran amine (BDA) into theright motor cortex as described in Li and Strittmatter, J. Neurosci.23:4219-27 (2003). In littermate WT mice, the prominent dorsal CST(dCST) is tightly bundled rostral to the lesion, and a few dorsolateralCST fibers are visible ipsilaterally. A small number of BDA-labeledshort collateral sprouts project into gray matter, particularly in theventral cord, but the sprouting is largely confined to the side of thecord contralateral to the tracer injection. However, the sectionsrostral to dorsal hemisection from injured sNogoR310 transgenic miceindicate a quite different BDA labeling pattern. A high density ofBDA-labeled CST fibers are observed outside of prominent dCST in all thetransgenic mice from line Tg08 or line Tg01. Ectopic fibers extendthroughout the gray matter area, and some fibers reach into lateral anddorsolateral white matter. Several fibers (4-12 sprouts per transversesection) are seen on the opposite side of the spinal cord (ipsilateralto the tracer injection site). Micro densitometric measurement of thecollateral sprouts indicates approximately a tenfold increase insprouting density in sNogoR310 transgenic mice. Examination ofparasagittal longitudinal sections from 1 to 4 mm rostral to the lesionreveals that dCST fibers extend a large number of branching sprouts intothe ventral gray matter area in sNogoR310 transgenic mice, in contrastto the littermate WT animals. Generally, the pattern and extent ofsprouting rostral to the lesion in transgenic mice are similar to thoseobserved in the mice treated systemically with Nogo receptor-1antagonist peptide NEP1-40 (Li and Strittmatter, J. Neurosci, 23:4219-27(2003)).

These results demonstrate that secreted sNogoR310 induces CST sproutingin the transgenic mice.

EXAMPLE 17 Regenerating CST Axons Bypass the Lesion Site into DistalSpinal Cord in sNogoR310 Transgenic Mice

We isolated spinal cord 4 mm rostral to and 4 mm caudal to the lesionsite (8 mm long in total) from transgenic mice and embedded it in aglutaraldehyde-polymerized albumin matrix, and cut parasagittally on avibratome (30 μm thick). We collected transverse sections (50 μm) fromthe spinal cord 5-7 mm rostral to and 5-7 mm caudal to the injury site.For sNogoR310-Fc injection experiments in rats, the spinal cordextending from 10 mm rostral to 10 mm caudal from the lesion site wascut parasaggitally (50 μm) on a vibrating microtome. Transverse sectionswere collected from the spinal cord 11-16 mm rostral to and 11-16 mmcaudal to the injury site. We incubated the sections withavidin-biotin-peroxidase complex and visualized the BDA tracer bynickel-enhanced diaminobenzidine HRP reaction (Grandpre, Nature417:547-551 (2002)). We processed some sections for serotoninimmunohistochemistry (anti-5-HT antibody) by indirect immunofluoresence.To visualize the lesion area, we double-stained some sections withantibodies directed against GFAP (Sigma®, St. Louis, Mo.). We mounted,dehydrated and covered the sections with mounting medium.

We tested whether the fibers induced by sNogoR310 expressed intransgenic mice after injury (see Example 16) cross the lesion area intothe caudal spinal cord to provide functional recovery.

Consecutive parasaggital sections across the injury site drawn in cameralucida display the overall distribution pattern of the regenerating CSTfibers a few millimeters from the lesion. Sections from WT mice show noCST fibers extending beyond the injury site. Similar sections fromsNogoR310 transgenic mice display numerous CST fibers that cross thetransection area and project into the distal gray and white matter areasin a highly branched pattern. Immediately rostral to hemisection, a highdensity of BDA-labeled CST sprouting originated from prominent dCSTprojects into the lesion area, but most CST sprouts failed to pass thetransection area where scar formation and tissue cavitation areprominent. A small but highly significant fraction of the regeneratingaxons bypass the lesion site through the remaining tissue bridges of theventral and ventrolateral gray and white matter. In addition, a few CSTfibers appear to cross the transection area itself via the lesioneddorsal and dorsolateral spinal cord into distal regions. In the vicinityof lesion, the course of regenerating fibers was typically tortuous andquite distinct from the normal straight fibers in the rostral CST.Collaterals and arborized fibers are most frequently seen in gray matterarea of distal spinal cord. The reconstructions demonstrate 5-15BDA-labeled regenerating fibers coursing in the rostral-caudal axis atany level 1-4 mm caudal to the lesion in each transgenic mouse. Fortransverse sections 5-7 mm caudal to dorsal hemisection, BDA-labeled CSTaxons are seen in both the gray matter and white matter areas in eachtransgenic mouse. The fiber counts for the transgenic mice indicateapproximately a similar number of BDA-labeled CST fibers to the proximallevels in the sagittal sections.

In addition to CST fibers, the other descending tracts, such asraphespinal fibers, also contribute to locomotor function in mice. Inthis mouse dorsal over-hemisection model, the transection injures amajority of the serotonergic fibers, decreasing the density of thesefibers by approximately 80% in the ventral horn. Analysis of totallength of serotonin fibers in the ventral horn of caudal spinal cordindicates a much greater number of these fibers in transgenic mice thanWT group, indicating that the growth-promoting effects of sNogoR310 intransgenic mice are not limited to one axon descending pathway.

EXAMPLE 18 Transgenic Expression of sNogoR310 Improves LocomotorRecovery

The CST axon tracing and serotonergic fiber analysis indicate that thesNogoR310 released from astrocytes in transgenic mice stimulatesextensive anatomical regeneration of injured descending axons in thespinal cord. We performed several behavioral tests as described inExample 12 to determine whether these regenerated fibers benefitfunctional recovery.

As assessed by the BBB test, the WT mice partially recover locomotorfunction during a 4-week period of survival. At 4 weeks post-injury,most WT mice recover a level characterized by consistent plantarstepping with consistent weight support, but they exhibit onlyoccasional to frequent forelimb-hindlimb coordination, with a rotationof predominant paw position when making initial contact with surface. Incontrast, the BBB scores of sNogoR310 transgenic mice from both linesTg08 and Tg01 are significantly higher than control group throughout the7-28 day observation period (FIGS. 13A and 13B). At 28 days afterinjury, most transgenic mice show consistent forelimb-hindlimbcoordination, and the predominant paw position is parallel to the body.

We employed two more behavioral tests to further characterize theperformance of sNogoR310 transgenic mice. First, we measured the maximalangle to which a board would be tilted without a mouse losing its gripwithin 5 sec. Before dorsal hemisection injury, both transgenic and WTmice can sustain their posture on board angled at 55 degrees. On days7-28 after injury, the sustainable angle is reduced in all mice, but theangles sustainable by the transgenic mice are significantly greater thanthose for the control group (FIG. 13C). In another behavioral test, miceclimbed a grid placed at a 45 degree angle to vertical and excursions ofthe hindlimbs below the plane of the grid were counted (Metz et al.,Brain Res. 883:165-177 (2000)). No mice made errors on this test duringthe pre-injury training. There are numerous foot fault errors with onlyminimal improvement in WT mice during the period 2-6 weeks post-injury.In contrast, the sNogoR310 transgenic mice exhibit a progressiveimprovement in grid climbing during this period, with the majority ofimprovement occurring between 1-3 weeks post-injury (FIG. 13D). Thus,transgenic mice secreting sNogoR310 from astrocytes exhibit CSTregeneration, raphespinal sprouting and improved motor function afterthoracic spinal hemisection.

EXAMPLE 19 Intrathecal Administration of sNogoR310-Fc Protein InducesCST Sprouting

As a second test of the growth-promoting benefit of soluble Nogoreceptor-1 after spinal trauma, we administered the purified proteinintrathecally.

We fused the ligand binding domain (27-310) of rat Nogo receptor-1 tothe rat IgG1 Fc domain to promote stability and purification. Wepurified protein from stably transfected CHO cells. This protein blocksNogo-66, MAG and myelin action in vitro, as shown previously for mousesNogoR310-Myc His (Fournier et al., J. Neurosci. 22:8876-8883 (2002);Liu et al., Science 297:1190-1193 (2002)). We delivered sNogoR310-Fcprotein intrathecally to rats with a mid-thoracic dorsal hemisectioninjury through an osmotic minipump. During a four-week survival periodafter injury, 1.2 mg sNogoR310-Fc protein was locally administered ineach rat. In rats receiving the vehicle treatment (1.2 mg rat IgG),sections rostral to hemisection display the tightly bundled prominentdorsal CST and very few ectopic BDA-labeled CST fibers above the lesionsite. Sections rostral to lesion from injured rats receivingsNogoR310-Fc protein exhibit a quite different pattern of labeling.Numerous ectopic fibers sprouting from the BDA-labeled CST are observedfrom transverse and parasagittal sections. In some cases, projectionscross from the dCST area near the midline to the circumference of thecord, becoming intermingled with the dorsolateral CST. The sproutingaxons extend through gray matter to a greater extent than white matter.A measure of ectopic sprouting fibers (≧100 μm in transverse sections,≧200 μm in sagittal sections) adjacent to the dCST reveals a greaterincrease in the sNogoR310-Fc-treated rats.

EXAMPLE 20 CST Axons Regenerate into Distal Spinal Cord in sNogoR310-FcTreated Rats

We deeply anaesthetized female Sprague-Dawley rats (190-250 g) andconducted laminectomies at spinal levels of T6-7, exposing the spinalcord. We cut the dorsal half of the spinal cord with a 30-gauge needleand a pair of microscissors to sever the dorsal parts of CSGT tracts,and assured the depth of the lesion (1.8 mm) by passing the sharp partof a number 11 blade across the dorsal half of the cord (Grandpre etal., Nature 417:547-551 (2002)). An osmotic minipump (Alzet® 2ML4, 2 mlvolume, 2.5 μl/h, 28 day delivery), which was filled with 1.2 mg rat IgGin PBS or 1.2 mg sNogoR310-Fc fusion protein in PBS, was sutured tomuscles under the skin on the back of the animals. A catheter connectedto the outlet of the minipump was inserted into the intrathecal space ofthe spinal cord at the T7-8 level through a small hole in the dura.

Nogo receptor-1 antagonist protein infusion induced extensive sproutingrostral to a rat hemisection, but a more critical issue is whether thesprouting CST fibers project to distal spinal cord and contribute tolocomotor recovery. Longitudinal sections across lesion site fromvehicle-treated rats display no detectable or a very small number ofBDA-labeled ventral CST fibers below the lesion level (Grandpre et al.,Nature 417:547-551 (2002); Weidner et al., Proc. Natl. Acad. Sci. USA98:3513-3518 (2001)). The similar sections from sNogoR310-Fc treatedrats demonstrate many BDA-labeled fibers bypass the transection site andproject to the caudal spinal cord largely through the bridging tissuesof the ventral and ventrolateral spinal cord. Immunostaining forastrocytic marker GFAP display that the extent of transection reacheddeeper than central canal area. Unlike the linear profile of rostralfibers in prominent dorsal CST, the regenerated CST fibers usuallyfollow a highly branching trajectory in the distal spinal cord,particularly in gray matter area. These fibers are detected in manyregions of spinal cord, but they are more easily seen in the centralpart and dorsal half of spinal cord throughout the spinal cord. Countsof CST fibers from sagittal sections indicate approximately 20BDA-labeled axons at 1-2 mm caudal to lesion and 15 traced axons at 7-8mm distal to lesion from each sNogoR310-Fc-treated rat.

Generally, the branching pattern of these fibers is similar to thatobserved from local NEP 1-40 peptide treated animals, but morecollateral branching in each sprout is seen from the sections treatedwith sNogoR310-Fc protein. A measure of the sprouts from distal spinalcord demonstrates that the total collateral length of each sprout insNogoR310-Fc-treated rats is twice as great as that from NEP 140-treatedanimals. The number of sprouts (≧200 μm in length) at 1-10 mm caudal tospinal cord in both Nogo receptor-1 antagonist-treated groups isapproximately 2040 times greater than control groups. More sprouts areseen from sNogoR310-Fc treated rats than local NEP 140 treatment (˜50vs. 25 sprouts/rat), but this difference is not statisticallysignificant (p=0.1713, t-test).

Regenerating CST axons are observed in transverse sections of spinalcord 11-15 mm caudal to hemisection in rats receiving sNogoR310-Fctreatment. These fibers are detected in both gray matter and whitematter of the spinal cord. The fibers detected in gray matter oftenexhibit more collateral branching than in white matter area. Incontrast, in transverse sections from vehicle-treated group, onlyoccasional BDA-labeled are seen in the ventral white matter area,consistent with the uninjured ventral CST axons. At this level of distalspinal cord, the average number of BDA-labeled CST fibers from both Nogoreceptor-1 antagonist-treated groups [sNogoR310-Fc and NEP 1-40] areapproximately 20-fold greater than vehicle-treated rats. Taken together,both Nogo receptor antagonists, sNogoR310-Fc protein and NEP 1-40peptide, result in dramatic CST axon regeneration in distal spinal cord,but the sprouting induced by the former exhibits a more highly branchedpattern.

EXAMPLE 21 Local sNogoR310-Fc Induces Sprouting Of Rubropinal andSerotonergic Axons in Injured Rat Spinal Cord

Fourteen days after hemisection, a burr hole was made on each side ofthe skull overlying the sensorimotor cortex of the lower limbs to traceCST fibers. The anterograde neuronal tracer BDA (10% in PBS, 3.5 μl percortex) was applied at seven injection sites at a depth of 1.5 mm fromdura on each side (Grandpre et al., Nature 417:547-551 (2002)). Forrubrospinal tract tracing in rats, the tracer BDA (1 μl; MW 10,000; 10%in PBS) was injected into red nucleus on the left side (5.8 mm posteriorto bregma, 0.7 mm lateral, 7.0 mm ventral to the skull surface). Twoweeks after BDA injection, these animals were perfused with PBS,followed by 4% paraformaldehyde, and tissue was collected for histology.

Repair of injured rubrospinal tract (RST) fibers contribute tofunctional improvements after spinal cord injury (Liu et al., J.Neurosci., 19:4370-4387 (1999)). The widespread distribution of Nogoreceptor-1 in CNS neurons (Wang et al., J. Neurosci. 22:5505-5515(2002)) makes it possible that inhibition of Nogo receptor-1 with itsantagonist may result in regrowth of RST axons after injury. To testeffects of sNogoR310-Fc on injured RST, the integrity of this pathwaywas traced by injecting BDA into left red nucleus. At the spinal cordlevel, RST fibers are normally located in dorsolateral white matter areaof spinal cord, and are transected by the dorsal hemisections of thisstudy. In transverse sections 11-15 mm rostral to lesion from controlrats, a small number of short BDA-labeled fibers are seen between theprominent RST and dorsal horn gray matter. Sections at same leveltreated with sNogoR310-Fc exhibit many linking fibers between the mainRST and dorsal horn gray matter. Transverse sections 11-15 mm distal toSCI, no BDA-labeled RST fibers in vehicle-treated rats. In contrast,sections at the same level receiving sNogoR310-Fc treatment display manyBDA-labeled RST fibers in both gray and white matter contralateral totracer injection. Some sprouts with a branching pattern are seen in thegray matter ipsilateral to BDA injection.

Ruphespinal spinal fibers were also examined in sNogoR310-Fc treatedspinal injured rats. Immunostaining demonstrates the density ofserotonergic fibers 11-15 mm rostral to lesion that is similar betweenvehicle and sNogoR310-Fc treated groups. In the sections 11-15 mm belowthe lesion, the seroton fibers in sNogoR310-Fc treated rats are twice asnumerous as those in the control group. These results demonstrate thatthe responsiveness to Nogo receptor-1 inhibition by sNogoR310-Fc proteinis not limited to CST fibers, and that the other descending tracts, suchas rubrospinal and serotonergic axons, are also responsive to Nogoreceptor-1 antagonism.

EXAMPLE 22 Local Treatment With sNogoR310-Fc Improves FunctionalRecovery in Rats

Intrathecal administration of sNogoR310-Fc protein stimulates axonregeneration in several descending pathways after traumatic spinal cordinjury. We tested whether the protein also improves functional recoveryin the injured spinal cord.

At 2 weeks after the hemisection, the locomotor BBB score invehicle-treated rats reaches a stable level of 12 (FIG. 14A). At 4 weeksafter lesion, most of controls (6 out of 7) have frequent-consistentweight-supported plantar steps and frequent-consistent forelimb-hindlimbcoordination, but they have a rotation of predominant paw position whenmaking initial contact with surface. In contrast, in rats receivingsNogoR310-Fc protein treatment, the locomotor score continues to improvebetween 24 weeks post-trauma. At 4 weeks after injury, all 9 of thesNogoR310-Fc treated animals had consistent forelimb-hindlimbcoordination and a parallel paw position at initial contact with thetesting surface.

Grid walking has been used to assess the deficits in descending finemotor control after spinal cord injury (Metz et al., Brain Res.883:165-177 (2000)). This performance requires forelimb hindlimbcoordination and voluntary movement control mediated by ventrolateral,corticospinal and rubrospinal fibers. During the pre-injury training,all the rats accurately place their hindlimbs on the grid bars. At 24weeks post-injury, control rats make 8-9 errors per session with onlyminimal improvement over time. In contrast, the rats treated withsNogoR310-Fc exhibit a progressive improvement on grid walking and makesignificant fewer errors (4-7/session on average). The majority of theimprovement occurs at 2-3 weeks after injury. Analysis of hindpawfootprints in control group displays that stride length is significantlydecreased and stance width is increased at 4 weeks post-hemisection,compared with uninjured rats or injured animals receiving sNogoR310-Fctreatment (FIG. 14C). Therefore, these multiple behavioral testsdemonstrate that blockade of Nogo receptor-1 function with localinjection of antagonist protein improves locomotor recovery afterinjury.

EXAMPLE 23 Binding of a Monoclonal Anti-NgR1 Antibody, 1D9, to SolubleRat Nogo Receptor 310 (SNgR310)

Structural analyses performed on the co-crystal complex of the 1D9 Faband a soluble fragment of rat NgR1 (srNgR310) shows that this antibodybinds near the junction of the N-terminus cap and leucine rich repeatdomain on rat NgR1. FIG. 15. 1D9 binds only to rat NgR1 and does notrecognize human or mouse NgR1, nor NgR2 and NgR3. For crystallization ofrat srNgR310-Fc with the 1D9 Fab, each macromolecule was cleaved withpapain and purified from the Fc portion and stored in 10 mM Hepes pH 7,50 mM NaCl. The complex was prepared at 80 μM each and mixed at avolumetric ratio of 2:1 with a reservoir solution consisting of 14%Peg3350, 0.4M Zinc Acetate, 0.1M Magnesium Chloride. The solution wasincubated at 20 C for 1 hr and centrifuged at 12,000×g for 3 minutes toremove precipitate. Crystals were grown by placing 3-5 μL of thesupernatant over wells containing 50% to 100% of the reservoir solutionat 20 C. Thin plate-like crystals grew over a period of 1 week at 20 C.The crystals were cryoprotected by quickly transferring into 0.2M ZincAcetate, 8% Peg3350, 25% Ethylene Glycol for 2 min and then frozen byquick transfer into liquid nitrogen.

Crystals approximately 10 μm thick diffracted to 3.2 Å at beamline X25at the National Synchrotron Light Source (Upton, N.Y.). Data processingwith the HKL program package v. 1.97 (Otwinowski, Z., and Minor, W.,Methods Enzymol 276:307-326 (1997)) revealed the crystals to belong to aP21212 space group and approximate cell dimensions a=90.6 Å, b=188.6 Å,c=125.5 Å, and α=β=γ=90, consistent with 2 Fab-NgR1 complexes perasymmetric unit.

The crystal structure was solved by utilizing information on multipleisomorphous replacement experiments on soaked crystals to identifycommon mercury sites bound to the NgR along with molecular replacement.The space group was identified by inspection of mercury and goldisomorphous difference patterson maps in which a consistent 5 sigma peakwas identified at the w=0 harker section. Molecular replacement withMOLREP (Vagin, A., and Teplyakov, A., J. Appl. Cryst. 30:1022-1025(1997)) utilizing a rat NgR homology model based on the human NgR1structure (pdb code 1OZN) (He, X. L. et al., Neuron 38:177 (2003)) and ahomology model for the 1D9 Fab led to placement of one NgR1, one Fab anda second NgR1 molecules with a resulting R-factor of 48% and cleardensity for the CDR regions of the Fab. The placement of the model wasconfirmed by mapping the mercury sites identified from differencepatterson maps onto equivalent positions on both NgR1 molecules nearAsp138 and His182. No additional Fab fragments have been clearlyidentified in the density. Refinement of the two NgR1 and 1 Fab usingCNX (Brunger, A. T. et al., Acta Crystallogr D Biol Crystallogr54:905-921 (1998)) to 3.2 Å resolution has proceeded with a currentR-factor of 42% and Rfree of 46%.

Table 8 shows the contacts between the 1D9 Fab and rat NGR1. Contacts inwhich atoms from the Fab are within 3.9A distance from atoms in rat NgR1are listed and those contacts that could form a hydrogen bond witheither the main chain or side chain have an associated asterisk (*).

TABLE 8 CDR L1 CDR L2 CDR L3 KSSQSLLNSRNRKNYLA WASTRES MQSYNLFT N31-1D9Y71-NgRI R33-1D9 Y71, D97*, A94-NgRI N34-1D9 S70*, Y71-NgRI CDR H1 CDRH2 CDR H3 GFSLSSYGVH VIWSGGNTHYNSALMS VGIYYEGAWFAY F27-1D9 S53-1D9P26-NgRI S79*-NgRI S28-1D9 G54-1D9 Y101-1D9 P26-NgRI R81-NgRI P73*,A74*, S76*, A50, V51*-NgRI S30-1D9 N56-1D9 Y102-1D9 A57*-NgRI Q78*-NgRIY71, P73, A50, V51*, L36*-NgRI S31-1D9 E103-1D9 G54-NgRI Q49, A50, V51,P52, A53*-NgRI Y32-1D9 G104-1D9 P26, P28- A53-NgRI NgRI *indicatesH-bond interactions

EXAMPLE 24 NgR RNAi Screening by Transient Transfection

Three RNAi constructs were designed against the human NogoR1 (FIG. 16A)transcript. RNAi-1 and RNAi-3 target the human NgR gene specifically.RNAi-2 was designed to target human, mouse, and rat NgR genes. Pairs ofDNA oligonucleotides were synthesized and constructed into a PolIIIpromoter based RNAi expression vector, pU6 that contained the human U6promotor, kar resistance gene, and a Pad cloning site. NogoR2m wasdesigned to carry two mismatches to the target sequence and serve as anegative control. The nucleotide sequences of these oligonucleotides areshown in FIG. 16B.

The RNAi constructs were screened initially by co-transfecting the humanNgR expression vector together with the RNAi expression plasmids(phU6NgR-RNAi-1, 2 and 3) in mouse L cells. Mouse L cells were plated in6 well culture plates and then transfected with control GFP reporterplasmid alone or with RNAi vector against GFP, pU6GFPRNAi (lanes 2 and3). The expression of GFP was monitored as a control for GFP genesilencing. Mouse L cells were transfected with 0.5, 1 or 2 μg of hNgRexpression vector (lanes 4-6). The DNA amount in each well was adjustedto a total of 4 μg DNA by adding pUC19 plasmid. The RNAi:target ratiowas 4:1. Five micrograms of hNgR vector was co-transfected with 2 μg NgRRNAi-1, 2, 3, or 2m plasmid (lanes 7-10). Forty eight hours posttransfection, cells were harvested in SDS loading buffer and subjectedto SDS-PAGE. The expression of hNgR was analyzed by western blot usingrabbit serum against polyclonal hNgR antibody R150 (panel A) ormonoclonal antibody 7E11 (panel B).

Effective NgR expression silencing was observed in phU6NgR-RNAi-1 and -2transfected cells on Western blot using NgR antibodies 7E11 (monoclonal)and R150 (rabbit polyclonal). The results are shown in FIG. 17. Theexpression of NgR was reduced to basal levels in NgR RNAi-1 and -2transfected cells. In contrast, NgR RNAi-3 did not show any significantreduction of NgR, which was similar to the control, mutant NgR RNAi-2m.Therefore, NgR RNAi-1 and -2 are effective in hNgR gene silencing.Transient transfection results demonstrated >90% inhibition of NgRexpression.

EXAMPLE 25 Human NGR Silencing Confirmation

Although the detected signals are specific to hNgR (only in hNgR cDNAtransfected cells) with two types of antibodies (FIG. 17), the apparentMW of the detected bands (˜50 kD) was lower than expected. While notbeing bound by theory, this is probably due to the altered glycosylationof human NgR in mouse L cells. In order to confirm the observation onNgR MW discrepancy, hNgR cDNA transfection was carried out again inhuman SKN cells and 293 cells using Lipofectin. Forty-eight hours posttransfection, cells were harvested in SDS loading buffer and subjectedto SDS-PAGE. The expression of NgR was detected by Western blot usingboth 7E11 and R150 as described above. No hNgR specific signal wasdetected in parental SKN or 293 cells and the apparent MW of hNgRdetected with R150 is of expected in both SKN and 293 cells, >65 kD(FIG. 18).

The RNAi mediated hNgR silencing was confirmed in SKN cells. SKN cellswere plated in 6 well culture plates and then transfected with controlGFP reporter plasmid alone or with RNAi vector against GFP, pU6GFPRNAi(lanes 2 and 3). The expression of GFP was monitored as a control forGFP gene silencing. SKN cells were transfected 2 μg of hNgR expressionvector (lane 4). DNA amount in each well was adjusted to total of 4 μgDNA by adding pUC19 plasmid DNA. 0.5 μg hNgR vector was co-transfectedwith 2 μg NgR RNAi-1, 2, 3 or 2m plasmid (lanes 5-8). Forty-eight hourspost transfection, cells were harvested in SDS loading buffer andsubjected to SDS-PAGE. The expression of hNgR was analyzed by westernblot using rabbit serum against hNgR R150. Again, greater than 90% NgRknockdown was demonstrated in all NgR RNAi-1 and -2, but less efficientin NgR RNAi-3 and -2m (FIG. 19).

EXAMPLE 26 NGR Knockdown in Neuroscreen Cells

NeuroScreen cells expressing NgR were obtained from Cellomics Inc. forNgR function analysis. In order to achieve stable NgR knockdown inNeuroScreen cells, all RNAi constructs were converted into lentiviralvectors in either beta-gal or GFP backbones. A schematic representationof the lentiviral vector is shown in FIG. 20. Lentiviral vectors weregenerated by transfection of 293 cells with packaging plasmids(Invitrogen). To construct the lentiviral vector for stable expressionof NGR1 RNAi, the RNAi cassettes consisting of the hU6 promoter thatdrives the expression of the hairpins (i.e., Nogo-1, Nogo-2, Nogo-2m,and Nogo-3) were excised from the phU6 vector (described in Example 24)by PacI digestion and cloned into the unique PacI site of SSM007plasmids. See, for e.g. methods described in Robinson et al., NatureGenetics 33:401-406 (2003). To track lentiviral vector transduction, aCBA-GFP expression cassette or CMV-LacZ expression cassette wereinserted into SSM007 plasmid at the XbaI site, and the resultantconstructs were termed SSM007-BFGW and SSM007-BFZW, respectively.

After converting all NgR1 RNAi constructs into SSM007-BFGW andSSM007-BFZW backbone, the vectors were co-transfected with packagingplasmids, pLP1, pLP2 and pLP/VSVG into FT293 cells for lentiviral vectorproduction (Viropower kit, Invitrogen). pLP1 is a 8889 bp construct thatcontains the HIV-1 gag/pol sequence and the rev response element (RRE)expressed from a CMV promoter and with a b-globin poly A; pLP2 is a 4180bp construct that expresses Rev from the RSV promoter and with an HIV-1poly A to terminate the transcript; pLP/VSVG is a 5821 bp plasmid andexpresses Vesicular stomatitis virus glycoprotein G from the CMVpromoter and beta-globin poly A.

Due to the self-limiting effect of RNAi interference to lentiviraltiter, all viral stock titers appeared lower than regular lentiviralvectors, in the range of 4-5×10⁵ transducing unit in the culture medium.NgR RNAi lentivectors (LV-NgR RNAi) were used to transduce NeuroScreencells at moi (multiplicity of infection) of 1. The transductionefficiency was about 1% as indicated by GFP expression orbeta-galatosidase staining.

Because NgR RNAi-2 was demonstrated to be effective in NgR silencing andit targets all human, mouse and rat NgR, LV-NgR RNAi-2 was chosen totransduce NeuroScreen cells. Transduced cells were cloned by limiteddilution in 96 well plates. Beta-galactosidase positive or GFP positiveclones were identified and expanded for further NgR expression analyses.

About 20 cloned cell lines were analyzed for NgR expression by Westernblot using 7E11 monoclonal antibody. A typical western blot results isshown in FIG. 21. GAPDH was used as control for loading normalization.The NgR expression in all clones was quantified by densitometry scanningof the NgR bands on the western blot and normalized to GAPDH levels. Theratio of NgR vs GAPDH was used to measure NgR expression levels. Of the12 clones screened, 11 of them decreased NgR expression (using thelowest NgR expressing clone in LV-GFP transduced cells, 1E9, asreference). In contrast, all 4 LV-GFP transduced clones have comparableNgR levels as the naive NeuroScreen cells. FIG. 22. These resultsdemonstrate that stable cell lines were established with reduced NgRexpression.

EXAMPLE 27 Functional Analysis of LV-NGR RNAi Cells

We selected four clones from LV-NgR RNAi transduced cells for functionanalyses. Using the NgR levels of naive NeuroScreen cells as reference,the NgR levels of these four clones are approximately 10% for 3c12b, 20%for 3c4b, 30% for 5d12 and 60% for 4a12 of the naive cells, respectively(FIG. 23).

EXAMPLE 28 Mutations of Monoclonal Anti-NgR1 Antibody, 1D9 AllowRecognition of Human NgR1

By computer modeling it was shown that mutations in the 1D9 antibodyallow recognition of human NgR1. N56 of the 1D9 heavy chain, viacomputer modeling, can be mutated to serine, glutamic acid, asparticacid, or glutamine to interact with R78 of the human NgR1. In addition,R33 of the light chain can be mutated via computer modeling to alanineor serine to avoid the electrostatic and steric clashes with R95 ofhuman NgR1.

EXAMPLE 29 Ecto-Domain of the Rat NgR1(27-310) Fused to a Rat IGG butnot Methylprednisolone Reversed the Neurite Outgrowth Inhibitory Effectof Myelin in Dorsal Root Ganglion Cells

In investigating combined treatment with methylprednisolone (MP) andNgR(310)ecto-Fc for spinal cord injury (SCI), we sought to verify thatthese reagents have independent mechanisms of action. Briefly, myelinwas dried overnight in poly-L-lysine-precoated plates (Becton Dickinson,Bedford, Mass., USA) at 80 or 400 ng/well (2.5 and 12.7 ng/mm²,respectively). Wells were then coated with 10 μg/ml laminin (Calbiochem,La Jolla, Calif., USA) for 1 hour at room temperature (22-24 C).Embryonic day 13 chick dorsal root ganglion neurons were dissociated andplated for 6-8 hours as previously described (GrandPre et al., 2000;Fournier et al., 2001). Neurons were treated with 8 μM NgR(310)ecto-Fcin the presence or absence of 10 μg/nl MP (Pharmacia, Kalamazoo, Mich.,USA) for the entire outgrowth period. Neurons were then fixed andstained with βIII tubulin antibody (Covance, Princeton, N.J., USA) andneurite outgrowth was quantified using an automated cellular imaging andanalysis system (Axon Instrument, Union City, Calif., USA). Neuriteoutgrowth per cell was normalized to the average of duplicate controlwells for each experiment (n=3). The activity of NgR(310)ecto-Fc isbased on its ability to reverse the inhibition of axon growth by myelin.FIGS. 24 A-B. In contrast, MP alone had no effect on neurite outgrowthfrom dorsal root ganglion neurons on a myelin substrate and the presenceof MP did not alter axon growth stimulation by NgR(310)ecto-Fc. Thesedata indicate that MP does not directly influence myelin-induced neuriteoutgrowth inhibition and that MP and NgR(310)ecto-Fc have independentactions. These in vitro data support the hypothesis that MP andNgR(310)ecto-Fc will enhance SCI recovery in a sequentially effectivemanner.

EXAMPLE 30 Ecto-Domain of the Rat NgR (27-310) Fused to a Rat IGG andMethylprednisolone Treatment had a Temporally Distinct Effect onFunctional Recovery After Spinal Cord Transection

Both MP and NgR(310)ecto-Fc treatments had a temporally distinct effecton functional recovery after spinal cord transection. Briefly; femaleLong Evans rats (7 weeks old; Charles River, Wilmington, Mass., USA)were anesthetized using 25 mg/kg midazolam i.p. (Abott Laboratories,Chicago, Ill., USA) and 2-3% fluothane (Baxter, Deerfield, Ill., USA) inO₂ and a dorsal laminectomy performed at spinal level T6 and T7. Generalanesthesia was maintained at 1.5-2% fluothane in O₂ A dorsal hemisectionwas performed completely interrupting the main dorsomedial and the minodorsplateral corticospinal tract (CST) components. A microscapel wasused to sterotaxically transect the cord at a depth of 1.8 mm from thesurface of the cord. Immediately after CST transection, an intrathecalcatheter was inserted into the subarachnoid space at T7 and connected toa primed mini-osmotic pump inserted into the subcutaneous space. Themini-osmotic pumps delivered rat IgG isotype control protein orphosphate-buffered saline (PBS) (5 mg/ml, n=8) or NgR(310)ecto-Fc (50μM, n=19) at a rate of 0.25 L/h. A cohort of NgR(310)ecto-Fc treatedrats (n=8) were also treated with MP (Pharmacia; 30 mg/kg iv) and aseparate cohort treated with MP alone (30 mg/kg iv) immediately afterinjury and again 4 and 8 hours later. Functional recovery was assessedusing the BBB open field scoring method (Basso et al., J. Neurotrauma12:1-21 (1995)) the following day and weekly thereafter. Control animalsrecovered hindlimb function over the course of the study reaching a meanBBB score of 12±0.87 after 4 weeks. Mean BBB scores for treated groupsat the same time-point were: MP, 14.9±0.23; NgR(310)ecto-Fc, 14.8±0.24and NgR(310)ecto-Fc plus MP, 15.63±0.18. All treatment groups showedimproved BBB scores compared with controls over the course of the study.P<0/05 vs control, two-way repeated measure ANOVA with Tukey's posthoctest. (FIG. 25A). A statistically significant increase in BBB score wasobserved in MP− and MP plus NgR(310)ecto-Fc-treated rats the day aftersurgery compared with control animals or animals treated withNgR(310)ecto-Fc alone. BBB score was significantly improved inMP-treated rats 2 days after SCI. P<0/05 vs control, two-way repeatedmeasure ANOVA with Tukey's posthoc test. (FIG. 25B). This observationindicated an early effect of MP treatment on recovery. Given this veryearly effect of MP, BBB scores were normalized to day 2 to subtract outthis early effect of MP (FIG. 25C) thus illustrating the much lateronset of effect of NgR(310)ecto-Fc. BBB scores normalized to day 2 forindividual animals illustrate a significant improvement in functionalrecovery in NgR(310)ecto-Fc-treated rats±MP 2, 3 and 4 weeks after SCI.P<0/05 vs control, two-way repeated measure ANOVA with Tukey's posthoctest. (FIG. 25C). In combining treatment group normalized BBB scoresabrogated the enhancing effect of MP on NgR(310)(ecto)-Fc treatmentillustrating that (i) in the combined treatment group the effect of MPoccurred early after SCI and (ii) by subtracting out this effect therate and extent of functional recovery in the combined treatment groupand the NgR(310)ecto-Fc group were identical and more pronounced than MPtreatment alone.

A discriminating point on the BBB score is a score of 14, correspondingto consistent weight supported plantar steps and consistenthindlimb-forelimb coordination. Frequency of consistent plantar steppingand hindlimb-forelimb coordination, illustrating the proportion of ratsin each group that attained a score of 14 or higher 3 and 4 weeks afterSCI. Accordingly, results were expressed as the frequency with whichrats attained a score of 14 or greater; 50% of control rats attained ascore of 14 or greater by 4 weeks after injury (FIG. 25D). Combinedtreatment with NgR(310)ecto-Fc and MP significantly improved the rate offunctional recovery. P<0/05 vs control Fischer exact test. All rats(100%) treated with NgR(310)ecto-Fc or MP or combined therapydemonstrated consistent plantar stepping and coordinated movement by 4weeks. Combination therapy increased the rate of recovery of coordinatedfunction as a significantly higher proportion of this treatment groupreached a score of 14 or greater by 3 weeks compared with controls oreither NgR(310)ecto-Fc or MP treatment alone (FIG. 25D). Improvedfunctional recovery was also demonstrated as significantly improved meanstride length in NgR(310)ecto-Fc and NgR(310)ecto-Fc plus MP-treatedgroups compared with controls (FIG. 25E). MP treatment alone did notsignificantly improve stride length measured 4 weeks after SCI. P<0/05,one-way ANOVA with Dunnett's posthoc test.

EXAMPLE 31 Ecto-Domain of the Rat NgR1 (27-310) Fused to a Rat IGG andMethylprednisolone Treatment Enhanced Axonal Plasticity/RegenerationAfter Spinal Cord Transection

Treatment with NgR(310)ecto-Fc or combined treatment with MP andNgR(310)ecto-Fc enhanced axonal plasticity/regeneration after spinalcord transection. Briefly, for histological tracing of the CSTs, 2 weeksafter CST transection animals were re-anesthetized and an incision madein the scalp. The area around the skin incision was injected with alocal anesthetic, the left sensorimotor cortex exposed via a craniotomyand 7 μL 10% biotin dextran amine (BDA; 10,000 MW; Molecular Probes,Eugene, Oreg., USA) in PBS injected using a nanoliter injector andmicro4 controller at 12 points 0-3.5 mm posterior to Bregma and 0-2.5 mmlateral to the midline at a depth of 1 mm below the surface of thecortex. In some instances, the CST was labeled bilaterally using thesame procedure.

At 28 days after CST transection, the rats were anesthetized withinactin (100-110 mg/kg i.p.) and transcardially perfused withheparinized saline (100 ml, 10 iu heparin) followed by 4%paraformaldehyde (150 ml). Spinal cords were removed, postfixed in 4%paraformaldehyde and then impregnated with 30% sucrose for 48 hours; 25mm lengths of spinal cord, 10 mm rostral and 15 mm caudal to thetransection site, were embedded in optimal cutting temperature compound(OCT) with transverse segments of cord taken 10-15 mm rostral and 15-20mm caudal to the lesion.

Frozen sections (50 μm) were serially cut and stained withstrepavidin-conjugated AlexaFluor-594 (1:200; Molecular Probes) tovisualize labeled CST axons. Axon counts were performed on transversesections taken 10 and 15 mm caudal to the transection site. Allmeasurements were performed blind. Every eighth section, i.e., sections400 μm apart, was counted for each animal at each level of the cord andthe values were expressed as mean number of axons per section.

Treatment with NgR(310)ecto-Fc or combined treatment with MP andNgR(310)ecto-Fc resulted in significantly greater numbers of biotindextran amine (BDA)-labeled axons counted 15 mm caudal to the injurysite (FIG. 26A). BDA-labeled axons appeared to sprout from both thedorsal columns into the dorsal horn gray matter and the spared ventralCST, projecting into the ventral gray matter. Axon counts in discreteregions of the cord revealed the largest increase in axon number in thegray matter. The largest increase in axon numbers was observed in thegray matter (GM) compared with ventral white matter (vWM) and dorsalwhite matter (dWM) (FIG. 26B). P<0/05, one-way ANOVA with Dunnett'sposthoc test. These data suggest that treatment with NgR(310)ecto-Fcwith or without MP promotes plasticity in the spinal cord after injury.

EXAMPLE 32 Combined Treatment with Ecto-Domain of the Rat NgR1 (27-310)Fused to a Rat IGG and Methylprednisolone Increased the Number of AxonalConnections Between Biotin Dextran Amine-Labeled Corticospinal TractFibers and Lumbar Motor Neurons

Anti-vesicular glutamate transporter 1 (vGLUT1) antibody (dilution1:2500) was used to stain for neuronal cell bodies and α- and γ-motorneurons in lamina 9 were identified by their size and morphology. Thenumber of axons contacting α- or γ-motor neurons was significantlyincreased in the MP+NgR(310)ecto-Fc-treated group compared with controlanimals, with the most marked and significant effect observed in animalsreceiving combined treatment with NgR(310)ecto-Fc and MP (FIG. 27).P<0/05, one-way ANOVA with Dunnett's posthoc test.

Biological Deposits

Hybridomas HB 7E11 (ATCC® accession No. PTA4587), HB 1H2 (ATCC®accession No. PTA4584), HB 3G5 (ATCC® accession No. PTA4586), HB 5B10(ATCC® accession No. PTA-4588) and HB 2F7 (ATCC®accession No. PTA4585)were deposited with the American Type Culture Collection (“ATCC®”),10801 University Boulevard, Manassas, Va. 20110-2209, USA, on Aug. 9,2002.

As those skilled in the art will appreciate, numerous changes andmodifications may be made to the preferred embodiments of the inventionwithout departing from the spirit of the invention. It is intended thatall such variations fall within the scope of the invention.

It is claimed that:
 1. A NogoR polypeptide variant comprising: asNogoR310 polypeptide comprising the amino acid sequence of residues27-310 of SEQ ID NO: 58; and an antibody fragment, crystalizable (Fc);wherein the Fc is joined to the sNogoR310 c-terminus; and wherein theNogoR polypeptide variant inhibits neurite outgrowth inhibition.
 2. TheNogoR variant of claim 1, wherein said Fc is an IgG Fc.
 3. A dimericpeptide comprising two NogoR variants of claim 1.